KR20190069322A - Systems including a cell analyzer coupled to a mass spectrometer and methods using the systems - Google Patents

Abstract

Specific configuration of a system including a cell analyzer and mass spectrometer is described. In some embodiments, the system can be used to determine both the cell phenotype or cell response and the amount of at least one elemental species in the cell. The phenotype or other cell characteristics and element content of each cell in the cell population can be determined and correlated.

Description

TECHNICAL FIELD [0001] The present invention relates to a system including a cell analyzer coupled to a mass spectrometer, and a method of using the system. BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

Cross reference of related application

The present application is related to and claims priority to U.S. Provisional Patent Application No. 62 / 596,811, filed December 9, 2017, and U.S. Patent Application Serial No. 16 / 209,130, issued December 4,

Technical field

The specific configurations described herein are directed to systems and methods that can differentiate cells and provide differentiated cells to a mass spectrometer. More specifically, the specific configurations described herein use coupled flow cytometry and mass spectrometry to correlate individual cell characteristics with elemental levels of individual cells.

A measure of the absorption of a substance by living cells is usually difficult to determine. In many cases, it is impossible to determine whether any one cell actually consumes a particular substance, or a substance to which the cell is exposed.

In one embodiment, the method comprises providing individual cells in a population of cells from a cell analyzer to a mass spectrometer to quantify the amount of at least a first element in the provided individual cells, And correlating the quantified amount of the first element with the measurement of individual cells from the cell analyzer.

In a particular embodiment, the method comprises quantifying the amount of the first element in each cell of a population of cells using a mass spectrometer, and correlating the quantified amount of the first element of each of the cells with individual cell measurements from the cell analyzer . In some embodiments, the method comprises determining the number of cells in a population of cells comprising the first element, using a quantified amount of the first element of each of the cells and individual cell measurements from the cell analyzer. In other cases, the method comprises determining the number of cells in a population of cells exhibiting the selected biological response, using a quantified amount of the first element of each of the cells and individual cell measurements from the cell analyzer. In some embodiments, the method comprises correlating the quantified amount of the first element with the cell size using the quantified amount of the first element of each cell and the individual cell measurement from the cell analyzer. In some embodiments, the method comprises quantifying at least a second element in each cell of the cell population.

In another embodiment, the method comprises constructing a population of cells with a single cell suspension and then providing the single cell suspension to a cell analyzer. In some embodiments, the method comprises constructing a cell analyzer to provide single cell eukaryotic cells to a mass spectrometer or single cell prokaryotic cells to a mass spectrometer.

In some embodiments, the method comprises constructing a cell analyzer as a flow cytometer, a fluorescence activated cell sorter, or a magnetic sorter. In a particular configuration, the method comprises configuring the mass spectrometer to include an inductively coupled plasma. In another embodiment, the method comprises separating a population of cells from another species using chromatography and then providing the population of cells to a cell analyzer. In some embodiments, the method comprises determining cell size as a measure of individual cells from a cell analyzer. In certain embodiments, the method comprises determining cell viability as a measure of individual cells from a cell analyzer. In some embodiments, the method comprises determining cell phenotype using a specific label as a measure of individual cells from a cell analyzer. In some embodiments, the method comprises determining cell health using a specific label as a measure of individual cells from a cell analyzer. In some embodiments, the method includes configuring the cell to include an average size of about 0.2 to 100 microns, configuring the cell analyzer as a flow cytometer, and configuring the mass spectrometer to include an inductively coupled plasma .

In certain embodiments, the method comprises separating the organelles from the cell population, providing the individual organelles from the separated organelles to the mass spectrometer from the cell analyzer to quantify the amount of the first element in the individual organelles provided, And correlating the quantified amount of the first element with a measure of the individual organotypic from the cell analyzer.

In some embodiments, the method comprises correlating a quantified amount of at least a first element in an individual cell provided using a processor with a measure of an individual cell from the cell analyzer.

In another embodiment, a method of measuring the uptake of a first metal agent into a cell comprises exposing the population of cells to a first metal agonist, determining the phenotype of individual cells in the population of cells using a cell analyzer, Quantifying the amount of the absorbed metal agonist into each of the provided individual cells by providing the analyte from the cell analyzer to a fluidly coupled mass spectrometer in the cell analyzer and quantifying the amount of the first metal agonist absorbed into each of the individual cells And correlating the amount with a determined phenotype of each of the individual cells.

In certain embodiments, the method comprises constructing the mass spectrometer to include an inductively coupled plasma. In another embodiment, the method comprises selecting a first metal agonist as a transition metal agent or metal agent comprising an atomic weight of from 2 to 285 amu of an element. In some embodiments, the method comprises selecting the transition metal agonist as a DNA replication inhibitor and selecting the cell population as a human cell. In certain embodiments, the method comprises quantifying the amount of cells in a population of cells that are resistant to the uptake of a transition metal agonist, using a quantified amount of individual intracellular metal agonists and a determined phenotype of each of the individual cells of the population of cells .

In some embodiments, the method comprises exposing the cell to a second metal agent, wherein the second metal agent comprises a different metal than the first metal agent, exposing the second metal agent to a second metal agent using a cell analyzer, Quantifying the amount of the second metal agent absorbed into each of the provided individual cells by providing the individual cells from the cell analyzer to a fluid coupled mass spectrometer in a cell analyzer, Correlating the quantified amount of the second metal agent absorbed with each of the determined phenotypes of each of the individual cells. In some embodiments, the method further comprises determining the amount of a cell population that is resistant to the uptake of the second metal agonist using the determined amount of each of the individual cells of the cell population and the quantified amount of the second metal agonist in the individual cell Lt; / RTI >

In another embodiment, the method comprises constructing a cell analyzer as one of a flow cytometer, a fluorescence activated cell sorter, and a magnetic classifier. In some embodiments, the method comprises determining at least one of cell size or cell viability as a determined phenotype of the cell. In some embodiments, the method comprises labeling the cell with a specific label to determine the phenotype of the cell.

In another embodiment, a system is described that is configured to correlate the amount of a first element of a cell with a cell phenotype. In some embodiments, the system comprises a cell analyzer configured to provide individual cells from a population of cells and to determine the cellular measurements of the provided individual cells, a mass spectrometer configured to receive individual cells fluidly coupled to the cell analyzer and provided from the cell analyzer, , Which mass spectrometer is configured to determine the amount of the first element present in the individual cells provided and the processor is configured to correlate the determined cell measurements of the provided individual cells with the amount of the first element present in the individual cells provided.

In certain embodiments, the cell analyzer is configured as a flow cytometer. In another embodiment, the mass spectrometer comprises an inductively coupled plasma. In some embodiments, the system comprises a cell-introducing device fluidly coupled to a flow cytometer, e.g., the cell-introducing device includes a loop configured to provide the cells with a linear flow to the flow cytometer. In some embodiments, the cell introduction device further comprises a syringe. In certain embodiments, the cell introduction device comprises an autosampler. In another embodiment, the cell analyzer is configured as a fluorescence activated cell sorter or a magnetic cell sorter.

In some embodiments, the system includes a sample introduction device fluidly coupled to each of a cell analyzer and a mass spectrometer, wherein the sample introduction device is configured to receive individual cells from the cell analyzer and to provide the individual cells received to the mass spectrometer do. In some embodiments, the sample introduction device comprises a spray chamber.

In certain embodiments, the mass spectrometer is configured to determine the amount of the first metal as the first element. In another embodiment, the mass spectrometer includes an ion source fluidly coupled to the spray chamber, a mass analyzer fluidly coupled to the ion source, and a detector fluidly coupled to the mass analyzer.

In some embodiments, the cell analyzer is configured to determine the cell size, and the processor is configured to correlate the determined cell size of the individual cells with the determined amount of the first element present in the provided individual cells.

In another embodiment, the cell analyzer is configured to determine cell viability, and the processor is configured to correlate the determined cell viability of the individual cells with the determined amount of the first element present in the provided individual cells.

In a further case, the cell analyzer is configured to determine the cell phenotype, and the processor is configured to correlate the determined cell phenotype of the individual cell with the determined amount of the first element present in the provided individual cell.

Additional aspects, embodiments, examples, features, and configurations are described in further detail below.

Certain specific configurations of the system and method are described with reference to the accompanying drawings, in which:
Figure 1 is a diagram of a cell analyzer fluidly coupled to a mass spectrometer in accordance with certain embodiments.
2 is a diagram of a flow cell analyzer fluidly coupled to a mass spectrometer in accordance with some embodiments.
Figure 3 is a diagram of a fluorescence activated cell sorter fluidly coupled to two mass spectrometers according to a particular configuration.
Figure 4 is a diagram of a system including a cell analyzer and a mass spectrometer in accordance with some embodiments.
5 is a diagram of one sample introducing device according to a particular embodiment.
6 is a diagram of a torch and induction coil configured to hold an inductively coupled plasma according to some embodiments.
7 is a view of a torch and plate electrode configured to hold an inductively coupled plasma according to a particular configuration.
Figure 8 is a view of a torch and induction coil comprising a radial fin in accordance with some embodiments.
Figure 9 illustrates a method that can be used to correlate cell analyzer measurements with mass spectrometer measurements in accordance with some embodiments.
Figure 10 illustrates a method that can be used to determine the cellular uptake of a metal-based agent in accordance with some embodiments.
11 is a diagram of a system including a flow loop configured to introduce a sample into a cell analyzer in accordance with some embodiments.
Figure 12 is a graph showing the correlation between cell size and intracellular metal mass in accordance with some embodiments.
Figure 13 is another graph showing the correlation between cell size and intracellular metal mass in accordance with some embodiments.
14A is a graph showing the correlation between live cells and metal mass in individual living cells according to some embodiments.
FIG. 14B is a graph showing the correlation between live cell / dead cells and individual intracellular metal mass according to some embodiments.
Figure 15 is a graph showing the correlation of different fluorescent markers and individual intracellular metal masses according to certain embodiments.
Those skilled in the art will appreciate that, in view of the benefits of the present application, the elements shown in the figures are provided for illustrative purposes only. Other components may also be present and the components of the drawings may also be rearranged as desired. Any data shown in the figures is provided for illustrative purposes only and is not intended to represent any possible data representation or relationship between the cellular response / phenotype and the amount of a particular element or metal in a cell.

The following various configurations refer to the use of coupled flow cytometry and mass spectrometry, although other techniques and systems may also be used in place of, or in conjunction with, flow cytometry or mass spectrometry. For example, the flow cytometry analyzer can be replaced with a fluorescence activated cell classifier, a magnetic cell classifier, a cell classifier capable of differentiating cells according to size, or other devices capable of differentiating cells with different phenotypes or different properties. Each of the differentiated cells may then be provided with a mass spectrometer for detecting and / or quantifying the level of one or more substances in each of the individual cells. In some embodiments, one or more elemental species in each of the cells may be detected and / or quantified. The amount of elemental species in each cell and the specific phenotype of the cell (s) may be correlated to provide useful information, for example, the level of absorption in each cell of the agent comprising the elemental species.

1, a block diagram of a system 100 including a cell analyzer 110 fluidly coupled to a mass spectrometer 120 is shown. The cells introduced into the cell analyzer 110 can be distinguished from each other based on one or more physical or chemical properties. For example, protein molecules on the cell surface can be used to specifically bind to one or more agents that may include a label. The label can be detected to distinguish between cells containing surface proteins and cells not containing surface proteins. Since each cell is detected and / or counted using cell analyzer 110, individual cells can be provided to elemental content, mass spectrometer 120, the elemental content of individual cells can be determined, 2, 3, 4 or more elemental species can be detected. The phenotype of each cell can then be correlated with the determined elemental content.

In some embodiments, the cell analyzer 110 may be configured as a flow cytometer. A brief description of the flow cytometer is shown in FIG. The flow cytometry analyzer includes flow cells 205, inlet 215 for sheath fluid, and outlet 220, including sample inlet 210. When the cell suspension 202 flows into the flow cell 205 through the sample inlet 210, the cis fluid is introduced into the inlet 215 and the cells are fluidically concentrated in the nozzle region 225. This concentration of cells can move each individual cell to flow cells 205, allowing each cell to be exposed to light from light source 250. When the cell suspension is labeled with a luminescent label, the label can emit or scatter light and this light can be detected by a detector 260 optically coupled to the light source 250. When the light is scattered, the front and side scattered light can be detected depending on the type and position of the detector 260. In some embodiments, there may be more than one kind of detector, so that both the front and side scattered light can be determined. For example, the forward scattered light can be detected by placing the detector in front of the beam, and the side scattered light can be detected by placing one or more detectors on the side of the flow cell 205. [ Each cell can flow through a flow cytometer to determine one or more cellular properties and each cell can be addressed or counted so that when the addressed cells are provided to a downstream mass spectrometer, Can be correlated with the cellular properties determined by the analyzer.

In some embodiments, the cells may be introduced into a cell analyzer using a syringe, autosampler or other device. The linear flow of the cells may be provided in a cell analyzer by constructing a cell analyzer having a flow loop or other suitable structure. Cells can be introduced from syringes, microtiter plates, test tubes, vessels or other devices that can be used to hold cells for a period of time.

In some embodiments, the population of cells may be labeled with one or more labels that are specific for a particular cell characteristic. For example, a cell can express a specific protein on its cell surface. Antibodies or other agents capable of specifically binding to cell characteristics may include a label, which can be measured using a flow cytometer. In some embodiments, the antibody may comprise a fluorescent label, the luminescence of which can be measured using a flow cytometer. In certain cases, the population of cells is mixed with or incubated with the labeled antibody in a cell suspension to allow the labeled antibody to bind to the cell population. Excessive labeling can be removed by centrifugation (or other technique), and the cell population can then be provided to a flow cytometer to determine whether each determined cell contains the labeled antibody. The various types of antibodies, labels and cells that can be used in the systems and methods described herein are discussed in further detail below.

In some embodiments, the cell analyzer can be configured as a fluorescence activated cell sorter (FACS). FACS can separate the cell suspension into two subgroups based on fluorescent labels. For example, a cell labeled with a fluorescent monoclonal antibody may be classified as a cell that does not have a fluorescent monoclonal antibody or a cell that contains a different fluorescent monoclonal antibody. Each cell can be detected using a light source similar to the light source shown in the flow cytometer of FIG. Referring to FIG. 3, when individual cells pass through flow cells through outlet 310, they may be provided to deflector device 320 in the form of electronically charged droplets (not shown). The charge applied to the droplet may depend on the detected fluorescence. The deflector device 320 can pull or push the charged cell droplet and can direct the droplet to one of the channels. The sorted cells may be provided in one of the mass spectrometers 330 and 340 to detect one or more element species in the individual cells.

In some embodiments, magnetic cell sorting may be used in place of FACS. In the cell sorting, one or more epitopes of the cell may specifically bind to an antibody labeled with a self-label, e.g., a magnetic nanoparticle label. The deflector device 320 of FIG. 3 may be replaced by a device configured to provide a magnetic field. Cells containing self-labeled antibodies can remain in the magnetic field, while cells that do not have self-labeled antibodies pass through the magnetic field. Cells remaining in the magnetic field can be provided in separate containers, and the elemental content of such cells can be measured using mass spectroscopy. As non-self-labeled cells pass through a magnetic field, such cells may be individually provided to a mass spectrometer to determine the elemental content of such cells. If desired, the magnetic label can be used to remove non-target cells, i.e., using negative charge selection, while the target cells pass through a magnetic field and can be provided to a downstream mass spectrometer. In some embodiments, the self-labeled antibody may be specific for the fluorophore present on the different antibodies. Self-labeled antibodies form complexes with antibodies containing fluorescent moieties to enable FACS or a self-cell classifier, or both. Alternatively, a typical antibody that specifically binds to the fluorophore may be used if a specifically labeled fluorescent antibody can be produced and a self-labeled antibody to a specific epitope is not available or readily produced. Common methods of producing fluorescent and self-labeled antibodies are discussed in further detail below.

In other embodiments, the agent or substance that is absorbed by the cell can comprise one or more radioactive isotopes that can be determined indirectly, e.g., using a scintillation count, using a cell analyzer, and can be assayed directly using a mass spectrometer . ≪ / RTI > Exemplary radioisotopes are described in further detail below.

In certain embodiments, the mass spectrometer coupled to the cell analyzer may have a number of different forms. A general view of a mass spectrometer system coupled to a cell analyzer is shown in FIG. The system 400 includes a cell analyzer 410 fluidly coupled to a sample introduction device 420. The sample introducing device 420 is fluidly coupled to the ion generating source 430. The ion source 430 is fluidly coupled to the mass analyzer 440. The mass analyzer is fluidly coupled to the detector 450, which may be integral or separate from the mass analyzer 440. The processor 460 may be electrically coupled to one or more components of the system 400 to control various subsystems. In some embodiments, the sample introduction device 410 may be omitted and the cells may be provided directly from the cell analyzer 410 to the ion source 430.

In a configuration in which a sample introduction device is present, the sample introduction device may be a nebulizer, an aerosolizer, a spray nozzle or head or other device capable of providing cells to the ion source 430. In some embodiments, the sample introduction device may be configured as a spray chamber as shown in FIG. The spray chamber 500 generally includes an outer chamber or tube 510 and an inner chamber or tube 520. The outer chamber 510 includes double makeup gas inlets 512 and 514 and a drain 518. The supplemental gas inlets 512, 514 are generally fluidly coupled to a common gas source, but different gases may be used if desired. Although not required, the supplemental gas inlet 512, 514 may be positioned toward the center or outlet end 513, although shown as being positioned adjacent the inlet end 511. The inner chamber or tube 520 is positioned adjacent to the nebulizer tip 505 and configured to provide supplemental gas flow to reduce or prevent cells from reversing and / or depositing from the inner chamber or tube 520 And may include two or more microchannels 522, The configuration and location of the inner chamber or tube 520 provides laminar flow in the areas 540 and 542, which serves to shield the inner surface of the outer chamber 510 from any droplet deposition. The tangential gas flow provided through the introduction of gas into the spray chamber 500 through the inlets 512, 514 may serve to select cells of a particular size range. The microchannels 522 and 524 in the inner chamber or tube 520 are also designed to shield the surface of the inner chamber or tube 520 from the cellular droplet deposition from gas flow from the supplemental gas inlet 512 and 514. In certain embodiments, the microchannels 522 and 524 may have different sizes, or different arrangements, such as, for example, the microchannels 522 and 524 may have the same size and / . In some cases, at least two, three, four, five or more separate microchannels may be present in the inner chamber or tube 520. The exact size, shape, and shape of the microchannels may vary, and each microchannel need not have the same size, shape, or shape. In some embodiments, microchannels of different diameters may be present in different radial planes along the longitudinal axis Ll of the inner tube to provide the desired shielding effect. Exemplary spray chambers are described, for example, in U.S. Provisional Patent Application No. 15 / 597,608, issued May 17, 2017, the entire disclosure of which is incorporated herein by reference for all purposes.

In certain embodiments, the exact dimensions of the spray chamber 500 may vary. In a particular configuration, the longitudinal length from the nebulizer tip 505 to the end of the spray chamber 500 may be from about 10 cm to about 15 cm, such as about 12 or 13 cm. The diameter of the outer tube 510 may vary from about 1 cm to about 5 cm, such as about 3 cm or 4 cm. The maximum diameter of the inner tube 520 may vary from about 0.5 cm to about 4 cm and the distance between the outer surface of the inner tube 520 and the retention surface of the outer tube 510 may be selected to provide the desired laminar flow rate For example, the distance may be from about 0.1 cm to about 0.75 cm. In certain embodiments, the inner tube 520 is generally shown as having an inner diameter that increases along the longitudinal axis of the outer chamber 510, but such a dimensional change is not required. A portion of the inner tube 510 may be " flat " or generally parallel to the longitudinal axis L1 to improve laminar flow, or, in an alternative configuration, a portion of the inner tube 520 may improve laminar flow Generally parallel to the surface of the outer tube 510, for at least some length. The inner diameter of the outer chamber decreases to some extent from the inlet end 511 toward the outlet end 513 and then decreases toward the outlet end 513 such that the inner diameter of the outer chamber 510 is greater than the outlet diameter at the inlet end 511, And smaller at end 513. The inner diameter of the outer chamber 510 may be maintained constant from the inlet end 511 toward the outlet end 513 or may increase from the inlet end 511 toward the outlet end 513. [ If desired, two or more different spray chambers, the same or different, may be fluidly coupled to each other to aid in the selection of individual cells.

In a particular configuration, the ion source 430 may have a number of different forms and is generally configured to ionize elemental species present in each individual cell. In some embodiments, the ion source may be an ion source of high temperature, e.g., an ion source with an average temperature above about 4000 Kelvin, for example, a direct current plasma, an inductively coupled plasma, an arc, a spark or other hot ion source. The exact source of ions used may vary depending on the particular element and / or cell being analyzed and exemplary sources of ions include ion sources such as metals, metalloids, and others capable of atomizing and / or ionizing the element species detected. And an ion source capable of atomizing and / or ionizing an inorganic species or an organic species. In another embodiment, the ion source may be an electron impact source, a chemical ion source, an electric field ion source, a tall source, such as a fast atom bombardment, field desorption, laser desorption, plasma desorption, thermal desorption, Or the like, a thermal spray or an electron spray ion source or other type of ion source.

In certain embodiments, the ion source can include one or more torches and one or more inductive devices. Specific components of the ion source are shown in Figures 6-8. Exemplary induction devices and torches are described, for example, in U.S. Patent Nos. 9,433,073 and 9,360,403, the entirety of which is incorporated herein by reference for all purposes. Referring to FIG. 6, an apparatus including a torch 610 in combination with an induction coil 620 is shown. The induction coil 620 is generally electrically coupled to a high frequency generator (not shown) to provide high frequency energy to the torch 610 and to maintain the inductively coupled plasma 650 within a portion of the torch 610. A sample introducing device (not shown) may introduce individual cells into the plasma 650 to ionize and / or atomize the elemental species present in the individual cells. The ionized and / or atomized element species may be detected in the torch using axial or radial detection, or provided to a downstream chamber or other device, e.g., a mass spectrometer, for detection or further selection and / or filtering. .

In an alternative configuration, the induction coil 620 of FIG. 6 may be replaced by one or more plate electrodes. 7, the first plate electrode 720 and the second plate electrode 721 are shown as including holes that can receive the torch 710. In one embodiment, For example, the torch 710 may be located within a portion of the inductive device including the plate electrodes 720, 721. A plasma or other ionization / atomization source 750, e.g., an inductively coupled plasma, can be maintained from the plates 720, 721 using torch 710 and induced energy. The high frequency generator 730 is electrically coupled to each of the plates 720 and 721. If desired, only a single plate electrode can be used instead. The sample introducing device may be used to introduce individual cells into the plasma 750 to ionize and / or atomize the intracellular species. Exemplary high frequency generators are described, for example, in U.S. Patent Nos. 4,629,940, 6,329,757, and 9,420,679.

In other configurations, an inductive device including one or more radial fins may be used instead in the methods and systems described herein. 8, an apparatus or system may include an induction coil 820 that includes at least one radial fin and a torch 810. As shown in FIG. Plasma or other ionization / atomization sources may be maintained (not shown), e.g., using torch 810 and inductive energy from radial pin guide 820. A high frequency generator (not shown) may be electrically coupled to the inductor 820 to supply high frequency energy into the torch 810. A sample introducing device (not shown) may be used to introduce the individual cells into the torch 810. Elemental species introduced into individual cells can be ionized or atomized and separated using a downstream mass spectrometer. In other cases, one or more capacitive devices, such as capacitive coils or capacitive plates, may be used in the ion source. Additional two or more inductive devices, capacitive devices, or other devices capable of providing energy into the torch to sustain an atomization / ion source, such as a plasma, may also be used.

In a particular embodiment, the mass analyzer 440 may have various forms depending on the sample properties, generally depending on the desired resolution, etc., and an exemplary mass analyzer is discussed further below. The detector 450 may be any suitable detector that can be used with conventional mass spectrometers, such as an electron multiplier, a Faraday cup, a coated photographic plate, a flash detector, etc., Lt; RTI ID = 0.0 > and / or < / RTI > Processor 460 generally includes software suitable for sample analysis to be introduced into a microprocessor and / or computer and system 400. If desired, one or more databases may be accessed by the processor 460 to determine the chemical identity of the species introduced into the system 400.

In certain embodiments, the mass analyzer 440 may have multiple forms depending on the resolution desired and the nature of the introduced sample. In a particular embodiment, the mass spectrometer may be a mass spectrometer, such as an inject mass analyzer, a magnetic sector analyzer (e.g. used in single and dual-integrated MS devices), a quadrupole mass analyzer, an ion capture analyzer (e.g., cyclotron, quadrupole ion capture) Analyzer, and other suitable mass spectrometers capable of separating and filtering (or both) elemental species having different mass-to-charge ratios. The mass spectrometer may comprise two or more different devices, such as a serial MS / MS device or a triple quadrupole device, arranged in series to select and / or identify ions received from the ion source 430.

In certain embodiments, the cell analyzer 410 is configured to determine the cell size, and the processor 460 is configured to correlate the determined cell size of the individual cells with the determined amount of the first element present in the individual cells. The cell size can be determined, for example, by measuring the forward light scattering of the cells using flow cytometry. The nature and / or amount of the first element may be determined using mass analyzer 440 and detector 450. The processor 460 may then correlate the size measurements from the cell analyzer 410 and the element measurements from the detector 450. [

In some embodiments, the cell analyzer 410 is configured to determine cell viability, and the processor 460 is configured to correlate the determined cell viability of the individual cells with the determined amount of the first element present in the individual cells. Cell viability can be determined, for example, by measuring the level of intracellular propidium iodide. Propidium iodide is normally blocked in live cells but can be absorbed in dead cells. Other materials and dyes may also be used to differentiate live cells and apoptotic cells.

In another embodiment, the cell analyzer 410 is configured to determine the cell phenotype, and the processor 460 is configured to correlate the determined cellular phenotype of the individual cell with a determined amount of the first element present in the individual cell. As referred to herein, a cellular phenotype can be determined using a labeled antibody specific for one or more antigens present in a cell.

In certain embodiments, the systems described herein can be used to correlate a determined amount of a first element within an individual cell with an individual cell phenotype. Referring to FIG. 9, the cell population may be provided to a cell analyzer at step 910. The cell analyzer may be configured to measure one or more physical or chemical properties at step 920. Individual cells from the cell analyzer can then be provided to the mass spectrometer at step 930. The mass spectrometer may determine the type and / or amount of one or more elements in step 940. The determined cellular nature may then be correlated with a determined amount of at least one element in step 950. [ For example, the phenotype of a cell and the amount of a specific element of the cell may be related to each other. The methodology shown in Figure 9 can be repeated for each cell in a cell population to correlate the quantified amount of each first element of the cell with individual cell measurements from the cell analyzer. Since all cells in a cell population can be determined, the method can be used to determine the number or percentage of cells in a population of cells comprising the first element or substance. Alternatively, the quantified amount of each first element or substance in the cell and individual cell measurements from the cell analyzer may be used to determine the number of cells in the cell population that represent the selected biological response, e.g., a particular antigen at the cell surface . In some embodiments, the cell analyzer can be used to determine the size of each cell in a population of cells, and the determined amount of the first element or substance in each cell can be correlated with a cell size measurement.

While the method refers to determining the first element (or other substance) in an individual cell, the same methodology may be used to determine the level of the second, third, or fourth element (or other substance) in each cell have. In some embodiments, the type and amount of two different elements present in a cell can be determined at the same time, which is co-ownership filed January 8, 2018, U.S. Patent No. 62 / 614,888, and Systems for Quantifying Two or More Analytes Using Mass Spectrometry ".

In certain embodiments, the methods described herein may preferably employ single cell suspensions of cells to increase the likelihood of being provided to a cell analyzer and / or mass spectrometer. Single cell suspensions may include allogeneic or heterogeneous cell populations and may include the same or different types of cells. Exemplary types of cells are discussed in more detail below and include both prokaryotic and eukaryotic cells. A number of different cell types and sizes may be used, and exemplary cell sizes may vary from about 0.2 microns to about 100 microns, for example, the average diameter of the cells may be from about 0.2 microns to about 100 microns. Larger or smaller cells may also be used. In addition, the organelles can be separated and introduced into the system described in the specification. Cells and cell populations may be subjected to one or more preparatory steps, such as chromatography, electrophoresis, cell size measurement, cell expression, labeling, culture, staining, or other steps, prior to introduction into a cell analyzer. As mentioned herein, a label is often used in combination with an agent that specifically binds to a site on a cell. Cells can also be treated with one or more dyes or agents that help to differentiate between cell size or live cell to cell death.

In some embodiments, the methods described herein can be used to determine or measure the absorption of an agent into a cell. For example, the absorption of the first metal agonist can be determined by measuring the level of the first metal in each of the cells. In some embodiments, the method comprises the steps of exposing a population of cells to a first metal agonist, determining the phenotype of individual cells in the population of cells using a cell analyzer, separating individual cells from the cell analyzer into a fluid- To an assay system to quantify the amount of metal agent absorbed into each of the provided individual cells and correlating the quantified amount of the first metal agent absorbed into each of the individual cells with a determined phenotype of each of the individual cells . A view of the above process is shown in Fig. At step 1005, the population of cells is exposed to a metal-based agonist. The free metal-based agonist can be removed from the cell by centrifugation, filtration or other steps. The population of cells exposed in step 1010 may be provided to a cell analyzer. In step 1020, the cell analyzer may perform one or more measurements, such as determining cell size, living or dead cell conditions, and the like. In step 1030, individual cells are provided to a mass spectrometer, which quantifies in step 1040 how much of the metal-based agent has been absorbed by the cells. The properties determined from the cell analyzer can then be correlated with the determined amount of metal from the mass spectrometer. Although metal-based agents have been described for illustrative purposes, they may be used as an indicator of how much of the element has been absorbed by the cell, instead of the metalloid or nonmetallic elements present in the agent.

In certain embodiments, the metal agonist may comprise an element species having an atomic weight of from 2 atomic mass units (amu) to 258 amu. In other cases, the first metal agent may comprise one or more transition metals or one or more transition metals. The metal or element selected for use may be intentionally chosen as a transition metal agent effective as a DNA replication inhibitor, such as cis-platin or other similar metal-containing agents. In some embodiments, the cellular amount of the cell population that is resistant to the absorption of the agent can be determined using a quantified amount of the individual intracellular agonist and a determined phenotype of each of the individual cells of the cell population.

In some embodiments, the cells can be exposed to a second metal agonist that includes a second metal agonist and a metal that is different from the first metal agonist. This exposure to two agents can occur sequentially or simultaneously. The phenotype of individual cells in a population of cells exposed to the second metal agonist can be determined using a cell analyzer. Individual cells from the cell analyzer can be provided to a mass spectrometer fluidly coupled to the cell analyzer to quantify the amount of second metal agent absorbed into each of the individual cells provided. The quantified amount of the second metal agonist absorbed into each individual cell may be correlated with the determined phenotype of each individual cell. In certain embodiments, the amount of cells in the population of cells that resist the uptake of the second metal agonist can be determined using a quantified amount of the second metal agonist in the individual cell and using the determined phenotype of each individual cell in the population of cells. The cell analyzer of Figures 9 and 10 may be a flow cytometer, a fluorescence activated cell sorter, a magnetic classifier or other suitable cell analyzer. Although not shown, a cell may be exposed to a labeled antibody or other substance capable of specifically binding to one or more sites on the cell.

In certain embodiments and as noted above, the systems and methods described herein may include or use a processor to control some aspect of the system or process. A processor may be part of, or associated with, a system or apparatus, such as a computer, laptop, mobile device, or the like. For example, the processor may be used to control the voltage provided to the mass analyzer and the introduction of the cis fluid into the cell analyzer, and / or may be used by the detector. This process can be performed automatically by the processor without requiring user intervention, or the user can enter parameters via the user interface. For example, a processor may use signal strength and fractional peaks with one or more calibration curves to determine its identity and determine how many specific elements are present in each individual cell. In a particular configuration, the processor may be configured to operate in one or more computer systems and / or to operate a system for controlling a common hardware circuit, e.g., a microprocessor, and / or for example a sample introducing device, ion generator, mass analyzer, Lt; RTI ID = 0.0 > software. In some embodiments, the detector itself includes its own processor, operating system, and other features capable of detecting various molecules. The processor may be integral to the system or may be on one or more accessory boards, printed circuit boards or computers electrically connected to the system components. A processor is typically electrically coupled to one or more memory units to receive data from other components of the system and to adjust the desired or desired various system parameters. Such a processor may be part of a general purpose computer, such as Unix, Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett-Packard PA-RISC processor, or any other type of processor. One or more of any type of computer system may be used in accordance with various embodiments of the technology. In addition, the system may be connected to a single computer or may be distributed among a plurality of computers attached by a communication network. It is to be appreciated that other functions may be performed, including network communications, and that the techniques are not limited to having any particular function or set of functions. Various aspects may be implemented as special software executed in a general purpose computer system. A computer system may include a processor coupled to one or more memory devices, such as a disk drive, memory or other device for storing data. Memory is typically used to store data such as the program, calibration curve, chemical or physical characteristics measured by a cell analyzer, and system operating. Components of a computer system may be coupled by interconnecting devices that may include one or more buses (e.g., between integrated components within the same machine) and / or network (e.g., between components residing on separate separate machines) have. Interconnects provide communication (e.g., signals, data, instructions) that is exchanged between components of the system. The computer system can typically receive and / or execute commands within a processing time, e.g., a few milliseconds, a few microseconds, or less, to allow for rapid control of the system. For example, computer control may be implemented to control sample introduction, flow rate, voltage provided to the components of the mass spectrometer, detector parameters, and the like. The processor is typically electrically coupled to a power source, which may be, for example, a direct current source, an alternating current source, a battery, a fuel cell or other power source or combination of power sources. Power can be shared by other components of the system. The system also includes one or more input devices, such as a keyboard, a mouse, a trackball, a microphone, a touch screen, a manual switch (e.g., an override switch) and one or more output devices such as a printing device, can do. The system may also include one or more communication interfaces for connecting the computer system to the communication network (additionally or alternatively to the communication device). The system may also include suitable circuitry for converting received signals from various electrical devices present in the system. Such circuitry may reside on a printed circuit board or may reside on a separate board or device electrically coupled to the printed circuit board via a suitable interface, for example, a Serial ATA interface, an ISA interface, a PCI interface, , Wi-Fi, Near Field Communication, or other wireless protocols and / or interfaces.

In certain embodiments, the storage systems used in the systems described herein generally include a computer readable and writable non-volatile recording medium, and the software code that may be used in such media may be stored in a program or program Lt; RTI ID = 0.0 > and / or < / RTI > The medium may be, for example, a hard disk, a solid state drive or a flash memory. The program or instruction executed by the processor may be located locally or remotely and may be retrieved by the processor by an interconnection mechanism, a communication network or any other desired means. In general, in operation, the processor allows data to be read from a non-volatile recording medium to another medium, allowing for faster access to information by the processor than the medium. Such memory is typically a volatile, random access memory such as a dynamic random access memory (DRAM) or a static memory (SRAM). Which may be located in a storage system or a memory system. The processor typically manipulates the data in the integrated circuit memory and then copies the data to the medium after processing is complete. Various mechanisms for managing the movement of data between a medium and an integrated circuit memory device are known, and the technology is not so limited. This technique is not limited to a specific memory system or storage system. In certain embodiments, the system may also include specially programmed special purpose hardware, such as an application-specific integrated circuit (ASIC) or a field programmable gate array (FPGA). Modes of technology may be implemented in software, hardware or firmware, or any combination thereof. Additionally, these methods, operations, systems, system components and components thereof may be implemented as part of the system described above or as an independent component. While a particular system is illustrated by way of example as a type of system in which various aspects of the technology may be practiced, it should be understood that such aspects are not limited to being implemented on the described system. Various aspects may be implemented on one or more systems having different architectures or components. Such a system may include a general purpose computer system programmable using advanced computer programming languages. The system may be implemented using specially programmed, special purpose hardware.

In such a system, the processor is generally a commercially available processor, such as a well-known Pentium class processor available from Intel Corporation. Many other processors are also commercially available. These processors typically operate one of the following operating systems: for example, Windows 95, Windows 98, Windows NT, Windows 2000 (Windows ME), Windows XP, Windows Vista, Windows 7, Windows 8 or Windows 10 operating system, Mac OS X available from Apple, such as Snow Leopard, Lion, Mountain Lion or other versions, Solaris operating system available from Sun Microsystems, or UNIX or Linux operating from various sources System. A number of different operating systems may be used, and in certain embodiments a simple instruction set or instruction may function as an operating system. A processor may also be designed as a quantum processor designed to perform one or more functions using one or more qubits.

In a particular embodiment, the processor and operating system can together define a platform in which an application program in a high level programming language can be written. It should be understood that this technique is not limited to a particular system platform, processor, operating system, or network. Also, in view of the benefits of the present invention, it will be apparent to those skilled in the art that such techniques are not limited to any particular programming language or computer system. It should also be appreciated that other suitable programming languages and other suitable systems may also be used. In certain embodiments, the hardware or software may be configured to implement a cognitive architecture, a neural network, or other suitable implementation. If desired, one or more portions of the computer system may be distributed over one or more computer systems coupled to the communication network. Such a computer system may also be a general purpose computer system. For example, various aspects may be distributed to one or more computer systems configured to provide services (e.g., servers) to one or more client computers or to perform the entire task as part of a distributed system. Various aspects may be performed in a client-server or multi-tier system that includes distributed components between one or more server systems that perform various functions in accordance with various embodiments. Such components may be executable, intermediate code (e.g., IL) or interpretive (e.g., Java) code, which communicates over a communications network (e.g., Internet) using a communications protocol . It is also to be understood that this technique is not limited to being performed on any particular system or group of systems. It should also be understood that this technique is not limited to any particular distributed architecture, network, or communication protocol.

In some cases, various embodiments may use object-oriented programming languages such as SQL, SmallTalk, Basic, Java, Javascript, PHP, C ++, Ada, Python, iOS / Swift, Ruby on Rails or C # Lt; / RTI > Other object-oriented programming languages can also be used. Alternatively, functional, scripting, and / or logical programming languages may be used. Various configurations may be implemented in an unprogrammed environment (e. G., A document formed in HTML, XML or other formats that render graphics-user interface (GUI) aspects or perform other functions when viewed in the window of a browser program) . The particular configuration may be implemented as a programmed or unprogrammed component, or any combination thereof. In some cases, the system includes a remote interface that resides on a mobile device, tablet, laptop computer, or other portable device capable of communicating over a wired or what interface and enabling the operation of the system remotely as desired .

In certain embodiments, the processor may include or access a database of information about atomic mass, mass-to-charge ratio, and element species that may include other common information. The instructions stored in the memory can in fact execute a software module or control routine for the system that can provide a controllable model of the system. The processor may use the information accessed from the database with one or a software module running on the processor to determine values of different components of the system, e.g., different cell analyzer parameters, different mass analyzer parameters, and so on. Upon receiving a control command and an output interface coupled to different system components of the system using the input interface, the processor can perform active control through the system. For example, the processor may control a detector, a sample introducing device, an ion source, a cell analyzer, a mass analyzer and other components of the system.

In certain embodiments, the exact cell type that may be used with the systems and methods described herein may vary. In some embodiments, the cell may be a prokaryotic cell. For example, drug detection methods using metal-based antibiotics can be performed by determining how many specific metal-based antibiotics are absorbed by individual bacterial cells and determining the phenotype of the cell, e.g., living or dead, Based drug candidate can be evaluated. If the cell is a bacterial cell, the bacterial cell can be one or more cells of the following: Aki gambling Te Solarium gyunmun (Acidobacteria), actinomycetes door (Actinobacteria), arithmetic gyunmun (Aquificae), armatimonadetes (Armatimonadetes), bacillus of doors (Bacteroidetes), kaldi Seri kummun (Caldiserica), chlamydia gyunmun (Chlamydiae), green sulfur bacteria statement (Chlorobi), nokman gyunmun (Chloroflexi), Chrissie came Ness gyunmun (Chrysiogenetes), cyanobacteria door (cyanobacteria), talcheol bacillus statements ( Deferribacteres), over aureus - the sequence gyunmun (Deinococcus - Thermus), Dick Tio posts by Moose gyunmun (Dictyoglomi), El Lucy micro emptying door (Elusimicrobia), blood Val night Hotel gyunmun (Fibrobacteres), posterior gyunmun (Firmicutes), Fu earthy Te Solarium gyunmun (Fusobacteria), Gem Marty Monastir gyunmun (Gemmatimonadetes), lentisphaerae (lentisphaerae), nitro Spira in (Nitrospirae), rich gyungang (Planctomycetes), proteobacteria door (Proteobac teria), naseonsanggyun door (Spirochaetes), synergistetes (Synergistetes), tenericutes door (Tenericutes), thermodesulfobacteria door (Thermodesulfobacteria), yeolpo gyungang (Thermotogae), or dainty gyungang (Verrucomicrobia). Roneun exemplary steel (class), the neck (order) and / or the (family) of bacterial cells that can be analyzed include, but are not limited to: Aki FIG bacteria (Acidobacteria), Blas Sat katel Liao (Blastocatellia) , alone pagae (Holophagae), Lou Val bacteria (Rubrobacteria), written in secret Ophelia (Thermoleophilia), nose Rio bacteria (Coriobacteriia), Ke Sidi microvias (Acidimicrobiia), Night lily Woodruff Astoria (Nitriliruptoria), liquid Tino bacteria (Actinobacteria) , aekkwi Pique ilseu (Aquificales), aekkwi Picard Seah (Aquificaceae), written hydrogenocarbonate Wu Ah (Hydrogenothermaceae), ilseu (Desulfurobacteriales), disulfonic furosemide bacteria Seah (Desulfurobacteriaceae), Thermo snowshoe dibak Termini (Thermosulfidibacter) in disulfonic furosemide bacteria, Pim Bree Mona Dia (Fimbriimonadia), Timothy Arma Nadia (Armatimonadia), chisso Nomo nade Tess (Chthonomonadetes), also written Mia (Rhodothermia), also sseome Ilseu (Rhodothermales), to neo Liao (Balneolia), to borneol Rails (Balneolales), ilseu (Sphingobacteriales) Saito wave Jia (Cytophagia), Saito wave Gale's (Cytophagales), sphingobacteria (Sphingobacteria), seuping securing Terry, Kitty hag Jia (Chitinophagia), Kitty old lady Gayle's (Chitinophagales), ilseu (Flavobacteriales) on the night Tero Dia (Bacteroidia), night teroyi Dales (Bacteroidales), Playa Bo bacteria (Flavobacteriia), Playa bobak Terry kaldi serika Seah ( Caldisericaceae), Cloud Mai diethoxy ilseu (Chlamydiales), Cloud Maida years old child (Chlamydiaceae), Candida tooth (Candidatus), Clavinova Cloud Maida years old child (Clavichlamydiaceae), para Cloud My Dales (Parachlamydiales), Cri Blossom Maida years old child (Criblamydiaceae ), para Cloud Maida Seah (Parachlamydiaceae), Siem Carney Asia (Simkaniaceae), wadeul called Seah (Waddliaceae), Candida Tooth Sickle piece Rama Dia (Candidatus Piscichlamydia), Candida tooth Cloud solution Tino Mardi Asia (Candidatus Actinochlamydiaceae), Candida Tooth Paril Cloud Mardi Asia (Candidatus Parilichlamydiaceae), Candida Tooth Rob also climb Mardi Asia (Candidatus Rhabdochlamydiaceae), Ignacio non-bacterial (Ignavibacteria), ilseu (Ignavibacteriales), Ignacio non-bacterial Seah (Ignavibacteriaceae), Ignacio thinning Te Leeum (Ignavibacterium), Melissa Duck Park Hotel (Melioribacter) in Ignacio thinning Terry, Chloe lobby (Chlorobea), ilseu the claw lobby (Chlorobiales), Chloe lobby Asia (Chlorobiaceae), not with the sword keulroriseu (Ancalochloris), chloro bar kyulryum (Chlorobaculum), chloro byum (Chlorobium), chloro-Herzegovina peton (Chloroherpeton), Carol Su as keulroriseu (Clathrochloris), Fellow dikton (Pelodictyon), Prosper sseko keulroriseu (Prosthecochloris), Thermo plexiglass ah (Thermoflexia), dehal rokokoyi Dia (Dehalococcoidia), wife Raleigh my (Anaerolineae), Ardennes Utica'll Ah (Ardenticatenia), Carl Dili my (Caldilineae), keute Tono bacteria (Ktedonobacteria), Thermo microvia (Thermomicrobia), chloro Plexiglass O (Chloroflexia), never.- Oh Genesee Tess (Chrysiogenetes), Chrissie Auger Nails (Chrysiogenales), Creative Make Jenna Seah (Chrysiogenaceae), croco Kale's (Chroococcales), croco Sidi Obsidian Dales (Chroococcidiopsidales), ilseu (Gloeobacterales) in ohbak Terry in writing, North SAT's scale (Nostocales), coming gelato Rails (Oscillatoriales), play right Cobb Sails (Pleurocapsales), RY rulri Nails (Spirulinales), cine chocolate's scale (Synechococcales), royalty reutae Three disks (Incertae sedis), to ferry bacterium Rails (Deferribacterale), to Perry Park Terra Seah (Deferribacteraceae), Day Noko Kale's (Deinococcales), Day-no Coca-year olds (Deinococcaceae), Tru Face Seah (Trueperaceae), written mail's (Thermales), written Wu Ah (Thermaceae), the mail's Dick toggle (Dictyoglomales), Dick toggle Rome Seah (Dictyoglomaceae), one Lucy microvias (Elusimicrobia), endo micro-vias (Endomicrobia), Blythe sat katel Ria (Blastocatellia) , Nice Kitty blood Lilia (Chitinispirillia),

Kitty Nibbionia ( Chitinivibrionia ), Fibrobacteria ( Fibrobacteria ), Bacillus (Bacilli), Bacillus ( Bacillales ), Lactobacillus root neck ( Lactobacillales ), Clostridium River Clostridia, Clostridiales,

Halla Nero Vail ( Halanaerobiales ), Natra Narrowvale ( Natranaerobiales ), Thermo wife Baptiste Riles (Thermoanaerobacterales), Ericipelotricia  (Erysipelotrichia), Ericipelotriechels ( Erysipelotrichales ), Nega TV (Negativicutes), Selenomonades ( Selenomonadales ), Sul Molysobacteria (Thermolithobacteria), Fusobacteria ( Fusobacteriia ), Fusobacterium (Fusobacteriales), Rept Totrich Asia ( Leptotrichiaceae ), Three Valle (Sebaldella), Sunica ( Sneathia ), Streptobacillus ( Streptobacillus ), Leptotrichia (Leptotrichia), Fusobacterium seah ( Fusobacteriaceae ), Setobacteria (Cetobacterium), Fusobacterium ( Fusobacterium ), Ilo Budapest ( Ilyobacter ), Propionic acid (Propionigenium), Cyclion Bakter ( Psychrilyobacter ), Logi Microvia (Longimicrobia), Gemma Timothy Dayz ( Gemmatimonadetes ), Oligosparia (Oligosphaeria), Lentis Paria ( Lentisphaeria ), Nightros Pyria (Nitrospiria), Knight Spirales ( Nitrospirales ), Nitro Spirasea (Nitrospiraceae), Fish Spa Seara ( Phycisphaerae ), Planktonisetacia (Planctomycetacia), Alpha proteobacteria ( Alphaproteobacteria ), Beta proteobacteria (Betaproteobacteria), Hydrogenophilaria ( Hydrogenophilalia ), Gamma proteo-bacteria (Gammaproteobacteria), Ashidi Ovaceli ( Acidithiobacillia ), Delta proteobacteria, Epsilon proteobacteria  And Oligoplexia (Epsilonproteobacteria and Oligoflexia ), Spyro Chattias ( Spirochaetia ), Brachyspirales, Brachi Spirasea ( Brachyspiraceae ), Brevinemates (Brevinematales), Blevine Matasea ( Brevinemataceae ), Leptospirosis (Leptospirales) Leptospirosa ( Leptospiraceae ), Spy Chase Tails (Spirochaetales), Borrelia Asia ( Borreliaceae ), Spy Chatha Seah (Spirochaetaceae), Sarpulina Seah ( Sarpulinaceae ), Senergistia (Synergistia),

Sinergy Stess ( Synergistales ), Siner Streptococcus ( Synergistaceae ), Mollicutes (Mollicutes), Heat deinking bacillus gate ( Thermodesulfobacteria ), Heat Deinking Bacteria (Thermodesulfobacterials), Heat deferred bacillus ( Thermodesulfobacteriaceae ), Fungi (Thermotogae), Kosomoto family ( Kosmotogales ), Kosomoto University ( Kosmotogaceae ), Mezzo Ashidi (Mesoaciditogales), Mesoaciditis ( Mesoaciditogaceae ), Petrotoman (Petrotogales), Petroto ( Petrotogaceae ), Thermal foliage ( Thermotogales ), Thermobacteria (Thermotogaceae), Perbivocuterium ( Fervidobacteriaceae ), Candidatus Epic Severo (Candidatus Epixenosoma ), Lentimonas ( Lentimonas ), Methyloacid (Methyloacida), Methyladidiumbromide ( Methylacidimicrobium ), Methyl acrylate (Methylacidiphilales), Spartobacteria ( Spartobacteria ), Opium Tutus River (Opitutae)or Umi ( Verrucomicrobiae ).The various genera and species within the steel, tree, and framework can be selected using the methods and systems described herein for analysis.

In other embodiments, the cell can be a eukaryotic cell and includes both " normal " eukaryotic cells present in properly functioning tissues and " abnormal " eukaryotic cells present in a cancerous or abnormal state. In addition, eukaryotic cells may be derived from protozoa, fungi, animals, plants, algae or other eukaryotic cells. The methods and systems may be particularly desirable for use in the investigation of fungal infections, cancer treatment efficacy, pesticide treatment efficacy, tissue repair status, and other conditions that may be monitored based on how much the material is in the cell.

If cells are fungal cells, fungal cells may be one or more cells of the following: blastocladiomycota (Blastocladiomycota), chytridiomycota (Chytridiomycota), taking gyunmun (Glomeromycota), US Cryptosporidium door (Microsporidia), neo potassium mas matrix gyunmun (Neocallimastigomycota), pair haekgyun ahgye (including incomplete gyunmun (Deuteromycota)) (Dikarya), ascus gyunmun (Ascomycota), pezizomycotina (Pezizomycotina), saccharomycotina (saccharomycotina), taphrinomycotina (Taphrinomycotina), basidiomycete Ryu (Basidiomycota) agaricomycotina (Agaricomycotina), pucciniomycotina (Pucciniomycotina), ustilaginomycotina (ustilaginomycotina), entomophthoromycotina (entomophthoromycotina), kickxellomycotina (Kickxellomycotina), mucoromycotina (Mucoromycotina), or zoopagomycotina (Zoopagomycotina) doors phyla and subphyla. Examples of fungal cells of the river, the neck and / or overwork, which may be analyzed include, but are not limited to: Blas toggle radio Wu test (Blastocladiomycetes), Blas Sat Cloud Madeira ilseu (Blastocladiales) Blas Sat Cloud di Asia (Blastocladiaceae) , Carthage Sahib Asia (Catenariaceae), koel our My theta Seah (Coelomomycetaceae), defendant further Mata Seah (Physodermataceae), Thoreau value tree Asia (Sorochytriaceae), cheat Reedy Oh, my three tests (Chytridiomycetes), Chi tree Dales (Chytridiales), Carol also cheat Rails (Cladochytriales), Rizzo blood Dales (Rhizophydiales), poly cheat Liege ilseu (Polychytriales), as a spinner gel My three tail switch (Spizellomycetales), Rizzo Montpellier Teasdale's (Rhizophlyctidales), to fire my three tail switch (Lobulomycetales), that our cheat Rails (Gromochytriales), meso cheat Rails (Mesochytriales), Shin cheat Rails (Synchytriales), poly wave Gale's (Polyphagales), Mo Noble Paris is also my three tests (Monoblepharidomycetes), mono Blake in Paris Dales (Monoblepharidales), Harpo Cheat Rails (Harpochytriales), hyaluronic Laura PD Oh, my three tests (Hyaloraphidiomycetes), Hebrews Alor Rafi Dales (Hyaloraphidiales), Article Romero Massena Tess (Glomeromycetes ) Oh chaos Four Rails (Archaeosporales), diver City Spokane Rails (Diversisporales), article Romer Rails (Glomerales), para posts Romer Rails (Paraglomerales), Cinema toffee tail's (Nematophytales), Metz Nico Bella (Metchnikovellea), Mech Nico Valley is cancer Pia Khan tidae (Metchnikovellida Amphiacanthidae), Mech Nico Valley for (Metchnikovellidae), micro Spokane below (Microsporea), Kou pick delridae (Cougourdellidae), pasil lease forage for (Facilisporidae), hetero Betsy particulate ridae (Heterovesiculidae), My sopori for (Myosporidae), nadel Spokane ridae (Nadelsporidae), neo-Nose Rana didae (Neonosemoidiidae), Ordos forage for (Ordosporidae), pseudo-year-old Marty (Pseudonosematidae), Tel Rome ripening period (Telomyxidae), Toxoplasma glue Gay for (Toxoglugeidae), tyubyul Reno-year-old Marty for (Tubulinosematidae), and the flow Pacifico (Haplophasea), cheat Reedy opsida (Chytridiopsida), cheat Reedy option period (Chytridiopsidae) , Bucks tehyu Saito gave for a didae (Buxtehudiidae), Enterprise (Enterocytozoonidae), version K for (Burkeidae), Heisei for (Hesseidae), a glue it (Glugeida), glue Gay for (Glugeidae), mop two (Gurleyidae), ense sold soil gave large (Encephalitozoonidae), Abel sports ridae (Abelsporidae), tube jetties for (Tuzetiidae), micro Phil versus (Microfilidae), Unicar are pleased versus (Unikaryonidae), Pacific (Dihaplophasea), Mei come below the flow wave to be die Let (Meiodihaplophasida), Pocatello do for cucumbers (Thelohanioidea), Pocatello do for (Thelohaniidae), du boss kwidae (Duboscqiidae), or for Seki (Janacekiidae), Tampere Zone (Pereziidae), the registry for forest Athos (Striatosporidae), tetracycline Los forage for (Cylindrosporidae), byure Nello two (Burenelloidea), byure Nelly vs. (Burenellidae), amble Rio Spokane two (Amblyosporoidea), amble Rios forage for (Amblyosporidae), di Sociedad Oda less flow wave Let (Dissociodihaplophasida), Nose Mato two (Nosematoidea), Nose vs. Marty (Nosematidae), Blake Tio vs. Sporting Reedy (Ichthyosporidiidae), kawoodo Spokane ridae (Caudosporidae), pseudo play for Stoke Pori (Pseudopleistophoridae), meura jeki for two prisoners Cooley course (Mrazekiidae Culicosporoidea), Cooley courses for forest (Culicosporidae), Cool Rico Spokane for Lely (Culicosporellidae), bone area Verbier (Golbergiidae), two soups rage Mr shi particulate Rio two (Spragueidae Ovavesiculoidea), Mr shi particulate ridae (Ovavesiculidae), tetra US Cree for (Tetramicridae), Rudy US cross Fora (Rudimicrospora), mini sports below (Minisporea), mini sports Florida (Minisporida), methoxy pitched Kovel below (Metchnikovellea), Menu pitched Kovel Florida (Metchnikovellida), (Polaroplasta) Plastic star as Paula, play Stoke forage for (Pleistophoridea), Place Topo Florida (Pleistophorida), di Castello below (Disporea), Unicar Rio Tia (Unikaryotia) dipole karioh Tia (Diplokaryotia), Neo Carly maseuti and my three tests (Neocallimastigomycetes), Neo Carly maseuti Gale's (Neocallimastigales), Neo Carly maseuti the Seah (Neocallimastigaceae), away Joe My Cortina (Pezizomycotina), are Sony Oh My three test (Arthoniomycetes), Connie My three five hundred forty-two beam test (Coniocybomycetes), Dottie to Oh My three tests (Dothideomycetes), Euro Tea Oh, my three tests (Eurotiomycetes), Geo-year-old Gloucester Mai Tess (Geoglossomycetes), La Bowl Benny Oh, my three tests (Laboulbeniomycetes), Recaro Noro My three tests (Lecanoromycetes), Leo tee Oh My three tests (Leotiomycetes), Rich no Mai-year-old Tess (Lichinomycetes), climb Billy Oh, my three tests (Orbiliomycetes) , Article away my three tests (Pezizomycetes), Oh My Broken leg three tests (Sordariomycetes), Mai-year-old Giles to test La's mail (Xylonomycetes Lahmiales), sense nodes climb on (Itchiclahmadion), assembly tree Dales (Triblidiales), Mai Cortina as Saccharomyces (Saccharomycotina), my three tests (Saccharomycetes as Saccharomyces), Tharp Reno My Cortina Ahkae duck crude My process (Taphrinomycotina Archaeorhizomyces), neo rekto my three tests (Neolectomycetes), peunyu linen styryl also my three tests (Pneumocystidomycetes), Shh irradiation Caro my three tests (Schizosaccharomycetes), Tharp Reno My three tests (Taphrinomycetes), are Tony Oh, my three tests (Arthoniomycetes), Connie five hundred forty-two Bo Mai three tests (Coniocybomycetes), Dottie to Oh My three tests (Dothideomycetes), Euro Tea Oh, my three tests (Eurotiomycetes), Geo Gloucester My three tests (Geoglossomycetes), La Bowl Benny Oh My Three tests (Laboulbeniomycetes), three Reka Noro My three tests (Lecanoromycetes), Leo tee Oh My test (Leotiomycetes), Rich no Mai-year-old Tess (Lichinomycetes), three climb Billy Oh, my test (Orbiliomycetes), away Joe My three tests (Pezizomycetes) , Oh My Broken leg three tests (Sordariomycetes), Giles as my three tests (Xylonomycetes), LA's mail (Lahmiales), Medo up Rails (Medeolariales), tree assembly Ilseu (Triblidiales), Mai a saccharide three tail switch (Saccharomycetales), ascorbyl the years old child (Ascoideaceae), three arms saccharide years old child (Cephaloascaceae), debari Oh, my theta years old child (Debaryomycetaceae), die poda Scar years old child (Dipodascaceae), endo Mai theta Seah (Endomycetaceae), lipoic Mai theta years old child (Lipomycetaceae), methoxy struck kowi Asia (Metschnikowiaceae), Resistencia Mai theta years old child (Phaffomycetaceae), pitch Asia (Pichiaceae), saccharide as MY theta years old child (Saccharomycetaceae), saccharide as MY coder years old child (Saccharomycodaceae ) My Cobb Let Seah (Saccharomycopsidaceae as Saccharomyces), tree chomo NASCAR Seah (Trichomonascaceae), Oh Kaori Joe My three tests (Archaeorhizomycetes), Neo rekto My three tests (Neolectomycetes), peunyu linen Stevenage also my three tests (Pneumocystidomycetes), My three tests to interrogate Saka (Schizosaccharomycetes), Tharp Reno My three tests (Taphrinomycetes), My dear Rico Laconia (Agaricomycotina), you push Maiko Tina (Pucciniomycotina), Wu Stila Gino Mai Cortina (Ustilaginomycotina), Walid Remy Oh, my three tests (Wallemiomycetes), Tre Melo My three tests (Tremellomycetes), the Cree My three tests (Dacrymycetes), Aga Rico My three tests (Agaricomycetes), Aga Rico steel beams My three tests (Agaricostilbomycetes), art plaque Tea Ilo My three tests (Atractiellomycetes), my age Clash Cool Tess (Classiculomycetes), Cryptococcus M. cola nose My three tests (Cryptomycocolacomycetes), cis Toba Sidi Oh My three test (Cystobasidiomycetes), micro boats Rio My three tests (Microbotryomycetes), Mixi Oh, my three tests (Mixiomycetes), push your Oh My three tests (Pucciniomycetes), tree tee Rocky Oh, my three tests (Tritirachiomycetes), exo-bar CD Oh, my three tests (Exobasidiomycetes) , aged Montserrat Oso Rails (Ceraceosorales), doah ssansil Rails (Doassansiales), entil Rome's tail (Entylomatales), exo-bashi Dales (Exobasidiales), If Fischer Rails (Georgefischeriales), micro-stroma tail switch (Microstromatales), teal lacy Els (Tilletiales), Wu Stila Gino My three tests (Ustilaginomycetes), right sheath tail switch (Urocystales), Wu Steel Large Nails (Ustilaginales), do Cecile Geo My three tests (Malasseziomycetes), do Cecile whether ilseu (Malassezioales), all nilri Ilo My three tests (Moniliellomycetes), all nilri El Rails (Moniliellales), bashi video Bolo My three tests (Basidiobolomycetes), Neo-being Saturday, my three tests (Neozygitomycetes), yen My three tests with Tomo neoplasm? (Entomophthoromycetes), ahselra Rails (Asellariales), Dima is Retail's (Dimargaritales), hapel Rails (Harpellales), kiksel Rails (Kickxellales), my age do not know Tyrrell Tess (Mortierellomycetes) , Tyrrell know Les (Mortierellales), my three tests (Mucoromycetes) in Temuco, Temuco Rails (Mucorales), or Endo and Nails (Endogonales). The various genera and species within the steel, tree, and framework can be selected using the methods and systems described herein for analysis.

If the cell is a plant cell, a plant cell may be at least one of the following: nematic toffee test (Nematophytes), chloro-phytase (Chlorophyta), arm a brush Rails (Palmophyllales), PRA genotyping blood years old child (Prasinophyceae), four Pro selmi escape Seah (Nephroselmidophyceae), Pseudomonas Scott Peel Dales (Pseudoscourfieldiales), minnow Mona escape years old child (Pyramimonadophyceae), to Umami del blood years old child (Mamiellophyceae), Scott Peel Dales (Scourfieldiales), page dino blood years old child (Pedinophyceae), chloro dendeuro blood Seah (Chlorodendrophyceae), TRE corrosion Sophie years old child (Trebouxiophyceae), crybaby blood years old child (Ulvophyceae), chloro blood years old child (Chlorophyceae), streptomycin phytase (Streptophyta), chloro keyboard phytase (Chlorokybophyta), meso stigmasterol Sat phytase (Mesostigmatophyta), keulrep sorbitan MIDI Opie other (Klebsormidiophyta), macaroni appetizer (Charophyta), caviar toss Parry Dales (Chaetosphaeridiales), nucleoside kaeto pita (Coleochaetophyta), if the swing Mato pita (Zygne matophyta), or Emporio pita pillar and the sub-pillar (Embryophyta phyla and subphyla). Including exemplary steel, tree, and and the genus of which can be analyzed cell, but are not limited to: nematic total Ruth (Nematothallus), Cosmo Klee or (Cosmochlaina), four Mato pita years old child (Nematophytaceae), nematic topless Lexus (Nematoplexus ), four Mata schedule Tomb (Nematasketum), Proto-suspended sheath (Prototaxites), eolbo blood years old child (Ulvophyceae), TRE diplopia operational years old child (Trebouxiophyceae), chloro blood years old child (Chlorophyceae), chloro dendeuro blood years old child (Chlorodendrophyceae), Mami Ilo blood Seah (Mamiellophyceae), four Pro selmi escape years old child (Nephroselmidophyceae), arm a brush-less (Palmophyllales), page dino blood years old child (Pedinophyceae), PRA genotyping blood years old child (Prasinophyceae), the shoe doses cor peel Dales (Pseudoscourfieldiales), minnow Mona escape Seah (Pyramimonadophyceae), SCOR peel Dales (Scourfieldiales), palmo Cloud truss (Palmoclathrus), a brush arm room (Palmophyllum), Verdi gel Ras (Verdigellas), Gino plastic nose Carlsbad (Prasinococc ales), PRA genotyping blood Seah Royalty reutae three disks (Prasinophyceae incertae sedis), the shoe doses cor peel Dales (Pseudoscourfieldiales), minnow Mona Dales (Pyramimonadales), four posel miss (Nephoselmis), peak no Coca years old child (Pycnococcaceae), SCOR pilda years old child (Scourfieldiaceae), Phedi grandma eggplant (Pedinomonas) , Li jeolteo (Resultor), Mar can Pio Pseudomonas (Marsupiomonas), the lock claw tree dione Tudor Bell Kula lithium (Chlorochtridion tuberculatum), chlorella's (Chlorellales), infrastructure support Olas (Prasiolales), Trail North Sails (Trebouxiales), Bree Obsidian Dales (Bryopsidales), climb applied Rails (Cladophorales), teahouse Cloud Dales (Dasycladales), Dale City to Ciel Baltic scanned only (Oltmannsiellopsidales), Scotty Bruno spa Rails (Scotinosphaerales), root rente pohil Rails (Trentepohliales), wool roteuri Kale's (Ulotrichales), wool bale's (Ulvales), kaeto pelti Dales (Chaetopeltidales), cache topology Rails (Chaetophorales), Cloud shown Mona Dales (Chlamydomonadales), the claw co-scale switch (Chlorococcales), the claw when stitcher Dales (Chlorocystidales), micro sports Rails (Microsporales), Oe Togo nielseu (Oedogoniales) , L ohpil Rails (Phaeophilales), sparrow flat Rails (Sphaeropleales), tetra sports Rails (Tetrasporales), chloro key booth (Chlorokybus), meso stigmasterol toffee years old child (Mesostigmatophyceae), Ent Cyano (Entransia), Hor MIDI Ella (Hormidiella), inter pilryum (Interfilum), keulrep sorbitan Medium (Klebsormidium), meso stigmasterol toffee years old child (Mesostigmatophyceae), keulrep sorbitan MIDI operational years old child (Klebsormidiophyceae), jig nematic toffee years old child (Zygnematophyceae), jig nematic tail des US Dales (ZygnematalesDesmidiales), drive blood years old child (Charophyceae), tea Rails (Charales), chloro keyboard blood years old child (Chlorokybophyceae), kolreoh car tail switch (Coleochaetales), poly chaeto Fora (Polychaetophora), while monohydrate L iridium (Chaetosphaeridium), kolreoh while toffee Seah (Coleochaetophyceae), jigs Cinema tail switch (Zygnematales), des US Dales (Desmidiales), Brighton OPA Estates (bryophytes), Similarly Tio pita (Marchantiophyta), Bra lo pita (Bryophyta), with no other ssose foreskin (Anthocerotophyta), Johor neo pie Top Cedar (Horneophytopsida), Trapani body OPA Estates (Tracheophytes), Lai Antonio pita (Rhyniophyta), Jos Terminus Philo pita (Zosterop hyllophyta), Rico Four video pita (Lycopodiophyta), Phyto-Pita (Trimerophytophyta), au Terry also pita (Pteridophyta), Stephen reuma topa Estates (Spermatophytes), au Terry Dos Fermat Saturday pita (Pteridospermatophyta), Pinot pita (Pinophyta) with a trimmer , Saika FIG phytase (Cycadophyta), Ginkgo phytase (Ginkgophyta), swing Sat phytase (Gnetophyta), or Magnolia Opie other (Magnoliophyta). Various species within the above tree, tree, vein, and genus can be selected using the methods and systems described herein for analysis.

In some embodiments, one or more analytes in a plant organ can be quantified using the methods and systems described herein. For example, plant organelles may include, but are not limited to, plant cell nuclei, nuclear membranes, nuclear membranes, vesicles, ribosomes, mitochondria, vacuoles, chloroplasts, cell membranes or cell walls. Plant organelles can be isolated from other materials in the cell and quantify analytes from isolated organelles.

When the cell is an animal cell, the animal cell may be an embryonic stem cell, adult stem cell, tissue-specific stem cell, mesenchymal stem cell, induced pluripotent stem cell, epithelial tissue cell, connective tissue cell, Lt; / RTI > Animal cells can be derived from ectoderm, endoderm or mesoderm. Ectodermal-derived cells include, but are not limited to, skin cells, pituitary gland cells, peripheral nervous system cells, neuroendocrine cells, teeth, ocular cells, central nervous system cells, pancreatic cells and pineal gland cells. Endoderm-derived cells include, but are not limited to, respiratory cells, gastric cells, intestinal cells, liver cells, gallbladder cells, exocrine pancreatic cells, Langerhans cells, thyroid cells and urinary epithelial cells. Examples of mesodermal-derived cells include osteochondroploid cells, fibroblasts, blood vessel cells, stromal cells, compacted cells, cells, interstitial cells, teloside, grapevine sites, cellulolytic cells, But are not limited to, peg cells, germ cells, hematopoietic stem cells, lymphocyte cells, bone marrow cells, endothelial progenitor cells, vascular endothelial progenitor cells, endothelial stem cells, myofibroblast / mesoangiocytes, .

In some embodiments, the animal cells are generally one or more other animal cells of a mammalian cell, such as a human cell, a dog cell, a horse cell, a cat cell, a bovine cell, and a mammalian cell: Artiodactyla, Carnivora, Cetacea, Chiroptera, Dermoptera, Edentata, Hyracoidae, Insectivora, Lagomorpha, Morasupilia, ), Monotremata, Perissodactyla, Pholidata, Pinnipedia, Primates, Proboscidea, Rodentia, Sirenia, and Kanchi (Turbulidentata). In some embodiments, the mammalian cell may be derived from a prosimian or a simian. In another embodiment, the mammalian cell may be derived from one or more of Adapiformes, Lemuriformes, Omomyiformes, and Tarsiiformes. In a further embodiment, the mammalian cell can be derived from one or more of Haplorhini or Simiiformes. In some embodiments, the mammalian cell may be derived from a human (Hominidae) or a human genome ( Homo ) , such as a human cell.

In some cases, cancerous animal cells may also be analyzed using the methods and systems described herein to assess the efficacy of treatment with a particular agent. The exact agonist may vary depending on the particular type of cancer to be treated, but the agent preferably causes a cancer cell to die in a certain way. Exemplary types of cancer from which cells can be analyzed include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), adrenocortical carcinoma, AIDS-associated cancerous sarcoma ), AIDS-related lymphoma (lymphoma), primary CNS lymphoma (lymphoma), anal cancer, appendix cancer, astrocytoma, abnormal teratoma / liver tumor, skin basal cell carcinoma, cholangiocarcinoma, bladder cancer, , Burkitt's lymphoma, cancerous adenoma, unknown primary carcinoma, cardiac tumor, abnormal teratoma / hepatic tumor, embryonal tumor, germ cell tumor, primary CNS lymphoma, cervical cancer, pediatric cancer, , Chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative cancer, colon cancer, craniopharyngioma, skin T-cell lymphoma, ductal carcinoma, endometrial cancer, uterine cancer), ependymoma, esophageal cancer, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, Gastrointestinal adenocarcinoma, , Germ cell tumor, pediatric germ cell tumor, testicular germ cell tumor, ovarian germ cell tumor, testicular cancer, pregnancy follicular disease, hair cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharynx Cancer of the head and neck, malignant fibrous histiocytosis and osteosarcoma of the bones, malignant melanoma, malignant fibrous histiocytosis, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant melanoma, Metastatic squamous cell carcinoma (head and neck cancer) with metastatic cancer, metastatic carcinoma with NUT gene change, oral cancer (head and neck cancer), metastatic squamous cell carcinoma Myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myelodysplastic syndrome, myelodysplastic syndrome, multiple myeloma / plasma cell neoplasia, Neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, lips and oral cavity cancer, oral cancer, osteosarcoma, and osteosarcoma. Pancreatic cancer, pancreatic neuroendocrine tumor, papilloma, papillary edema and nasal carcinoma, parathyroid cancer, penile cancer, pancreatic cancer (head and neck cancer), chromaffin cell tumor, Pituitary tumor, plasma cell neoplasm / multiple myeloma, pleural lung blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, (Sarcoma), skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, Lymphoma, pituitary cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid carcinoma, transitional cell carcinoma of the pelvic and ureter, metastatic carcinoma of the stomach (gastrointestinal cancer), T-cell lymphoma, Ureter and kidney pelvic cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, angioma, external tingling cancer, Wilms' tumor and other forms of cancer.

In some cases, the organelle's organelles can be isolated from other elements of the animal's cells, and then the organelle phenotype and elemental content of animal organelles can be quantified using the methods and systems described herein. The organelle phenotype (or the biological response of the organelle) can then be correlated with the determined element species. For example, isolated organelles include, but are not limited to, cell nuclei, nuclear membranes, microtubules, microfibers, endoplasmic reticulum, sarcoplasmic reticulum, ribosomes, mitochondria, vacuoles, lysosomes, But are not limited thereto.

In some embodiments, the methods and systems described herein can also be used to analyze viruses that may be present inside or outside the cell or both. For example, metal-based anti-viral efficacy binding to viral protein coat can be evaluated using the methods and systems described herein. When the virus is analyzed, the virus can be, for example, a double stranded DNA virus, a single stranded DNA virus, a double stranded RNA virus, a positive directed single stranded RNA virus, a negative directed single stranded RNA virus, a single stranded RNA- Virus) or double-stranded DNA reverse transcriptase virus. A variety of specific viruses include, but are not limited to: Paphos Bar bayireoseugwa, adenovirus bayireoseugwa, herpesviridae, herpes virus Thursday, ascorbic bayireoseugwa cancer pyulra bayireoseugwa, asparagus bayireoseugwa, as bayireoseugwa, pusel bayireoseugwa, glow fire to Bakool bayireoseugwa, beating bayireoseugwa, Hite Rosa bayireoseugwa, yirido bayireoseugwa, Lippo Mr. Riggs bayireoseugwa, Nima bayireoseugwa, Fox bayireoseugwa, Tech tee bayireoseugwa, corticosteroids bayireoseugwa, Sulfolobus, kawoodo viral throat, corticosteroids bayireoseugwa, Tech tee bayireoseugwa, Riga Men viral throat cancer Pula bayireoseugwa, non-Kau is bayireoseugwa, Cloud bar bayireoseugwa, pusel to bayireoseugwa, global bayireoseugwa fire, beating bayireoseugwa, Turi bayireoseugwa, ascorbic virus, baculovirus, Hite bayireoseugwa Rosa, Lido bayireoseugwa, poly and out virus, Mimi bayireoseugwa, Marseille Ile virus, MB virus, village virus by a phage, Sputnik by a phage, Nima bayireoseugwa, pico and out bayireoseugwa, play up Four virus, plasma bayireoseugwa, Pandora bayireoseugwa, Gavel and out virus, Ridge audio viruses, live Termini pro virus repositories in the spa bayireoseugwa, ah Nello bayireoseugwa, beads or bayireoseugwa, unsealing nose bayireoseugwa, Gemini bayireoseugwa, my grandma bayireoseugwa, Eno bayireoseugwa micro bayireoseugwa, nano bayireoseugwa, parvovirus bayireoseugwa, Spira bayireoseugwa, ahmalga bayireoseugwa, builder or bayireoseugwa, Cri small bayireoseugwa, Sisto bayireoseugwa, endo reuna bayireoseugwa, hypo bayireoseugwa, Mega builder or bayireoseugwa, Parr entity bayireoseugwa, Pico builder or bayireoseugwa, Choir laundry virus Leo bayireoseugwa, Totti bayireoseugwa, nidovirales blood corset or viruses neck, tie all viruses neck mononegavirales, Bor and bayireoseugwa, Philo bayireoseugwa, My Mona bayireoseugwa, Mao US bayireoseugwa, para mikso bayireoseugwa, pneumophila bayireoseugwa, Rob FIG bayireoseugwa, line bayireoseugwa, anpe virus, aryl virus, chenti virus, keureo star virus, West Lee virus, beonya virus neck, Blow bayireoseugwa, coat bayireoseugwa, hanta bayireoseugwa, John bayireoseugwa, the age bayireoseugwa, Perry beonya bayireoseugwa, fasteners do bayireoseugwa , page bayireoseugwa sister, sat bayireoseugwa Spokane Arena bayireoseugwa, Opio en Provence bayireoseugwa, ortho mikso bayireoseugwa, telta viruses, other European Astrid Virus, alpha retrovirus , avian leukemia virus; Rous sarcoma virus, retroviruses, beta, mouse mammary tumor virus, gamma retrovirus, murine leukemia virus, feline leukemia virus, bovine leukemia virus, human T- lymphoid friendly viruses, retroviruses Epsilon, corneal alum dermal sarcoma virus, lentivirus, human Immunodeficiency virus 1, monkey and cats immunodeficiency virus, spuma virus , monkey foamy virus, ortho retroviral subfamily , spuma retroviral subfamily , meta virus , pseudoviridae , retrovirus , hepadinaviridae , or cowl remyviridae . Various species within the above tree, tree, vein, and genus can be selected using the methods and systems described herein for analysis.

In a particular configuration and as noted herein, a cell analyzer can detect one or more markers present on an antibody that specifically bind to a site on a particular cell. Antibodies are polypeptides generally comprising a plurality of amino acids linked together through peptide bonds. The antibody may comprise an immunoglobin chain or fragment thereof comprising at least one immunoglobulin variable domain sequence. The term antibody includes, for example, monoclonal antibodies (including full length antibodies with immunoglobulin Fc regions), polyclonal antibodies and other polypeptides that are capable of specifically binding to one or more moieties of a cell. In one embodiment, the antibody molecule comprises a full length antibody, or a full length immunoglobin chain. In another embodiment, the antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. The amino acid of the antibody may be natural or synthetic and includes both amino and acidic functional groups and may be included in the polymer of the naturally occurring amino acid. Exemplary amino acids include naturally occurring amino acids; Analogs, derivatives and analogs thereof; Amino acid analogs having modified side chains; And all of the abovementioned stereoisomers. Both D- or L-optical isomers of amino acids and peptide mimics may be used. The term antibody also includes intact molecules as well as functional fragments thereof. The constant region of the antibody may be modified to alter the properties of the antibody (e.g., to increase or decrease one or more of the following characteristics: Fc receptor binding capacity, antibody glucosylation, number of cysteine residues, effector cell function, or complement function) , E. G., Mutated.

If desired, the antibody may be generated using conservative amino acid residue substitutions, wherein the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains will be selected by those skilled in the art in view of the benefits of the present invention. Such families include amino acids with basic side chains (e.g., acid, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine , Cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan , Histidine). The exact length of the antibody can vary as desired. In some cases, the synthetic polypeptides can bind to each other to form antibodies. The antibody may be linear or branched, may comprise modified amino acids, and may have discontinuities due to non-amino acids. Antibodies can be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation using label or label components. The polypeptide may be isolated from a natural source, produced by recombinant techniques from a eukaryotic or prokaryotic host, or may be the product of a synthetic procedure.

Antibodies can be expressed in a host system by inserting a suitable nucleic acid sequence into the host organism. The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence" and "polynucleotide" are used interchangeably. Refers to a deoxyribonucleotide or ribonucleotide, or analog thereof, as a polymeric form of the nucleotides of generally any length. The polynucleotide may be single-stranded or double-stranded, and the single-stranded strand may be a coding strand or a non-coding (antisense) strand. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs. Sequences of nucleotides may be terminated by non-nucleotide components. The polynucleotide may be further modified after polymerization by conjugation with the labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, which is not naturally occurring or is linked to another polynucleotide in a non-native sequence.

The antibody may be isolated from the host or other expression or production organism. As used herein, the term " isolated " refers to the removal of a substance from its origin or from its natural environment (e.g., the natural environment if it occurs naturally). For example, naturally occurring polynucleotides Or polypeptides are not separated, but the same polynucleotides or polypeptides separated by human intervention from some or all of the substances co-present in the natural system are isolated. Such polynucleotides may be part of a vector, and / Or such polynucleotides or polypeptides may be part of a composition, and such vectors or compositions may also be isolated in that they are not part of the environment found in nature.

In one embodiment, the antibody molecule binds to an animal cell, such as a mammalian cell. For example, the antibody molecule specifically binds to an epitope, e. G., A linear or stereomorphic epitope of mammalian cells, e. G., An epitope as described herein. In some embodiments, the antibody molecule binds to one or more extracellular Ig-like domains, such as a first, second, third or fourth extracellular Ig-like domain of a specific epitope present in a mammalian cell.

In one embodiment, the antibody molecule is a single specific antibody molecule and binds to a single epitope, e. G., A single specific antibody molecule having a plurality of immunoglobulin variable domain sequences, each binding to the same epitope.

In another embodiment, the antibody molecule is a multispecific antibody molecule, eg, comprising a plurality of immunoglobulin variable domain sequences, wherein the plurality of first immunoglobulin variable domain sequences have binding specificity for the first epitope, The plurality of second immunoglobulin variable domain sequences have binding specificities for the second epitope. In one embodiment, the first and second epitopes are on the same antigen, e. G., The same protein (or a subunit of a multisp protein). In other cases, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In another embodiment, the first and second epitopes are present in different antigens, such as different proteins (or different subunits of a multisp protein). In a further embodiment, the multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a triple-specific antibody molecule, or a quadruple-specific antibody molecule.

In some embodiments, the multispecific antibody molecule may be a bispecific antibody molecule. Bispecific antibodies have specificity for two or fewer antigens or two binding sites. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for the first epitope and a second immunoglobulin variable domain sequence having binding specificity for the second epitope. In another embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or a subunit of a multisp protein). In one embodiment, the first and second epitopes overlap. In yet another embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are present in different antigens, such as different proteins (or different subunits of a multisp protein). In another embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain having a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for the first epitope and a binding specificity for the second epitope Sequence. In other cases, the bispecific antibody molecule comprises a half-antibody having binding specificity for the first epitope and a half-antibody having binding specificity for the second epitope. In a further embodiment, the bispecific antibody molecule comprises a half-antibody, or fragment thereof, having binding specificity for the first epitope, and a half-antibody, or fragment thereof, having binding specificity for the second epitope. In one embodiment, the bispecific antibody molecule comprises a single-chain variable fragment (scFv), or fragment thereof, having binding specificity for the first epitope and scFv, or fragment thereof, binding specificity for the second epitope .

In another configuration, the antibody molecule comprises a diabody, and a single-chain molecule, as well as an antigen-binding fragment (e.g., Fab, F (ab ') ₂ , and Fv) of the antibody. For example, the antibody molecule may comprise a heavy chain variable domain sequence (abbreviated herein as VH) and a light chain (L) variable domain sequence (abbreviated herein as VL). In one embodiment, the antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, the antibody molecule comprises two heavy (H) variable domain sequences, and two light chains (L) comprises a variable domain sequence, the two antigen binding sites, such as Fab, Fab ', F (ab ') 2, Fc, Fd, Fd, Fv, single chain antibodies (e.g. scFv), single variable domain antibodies, Dabs (divalent and bispecific), and chimeric , Which can be generated by transformation of newly synthesized antibodies using whole antibodies or recombinant DNA techniques. Such a functional antibody fragment generally retains the ability to selectively bind to its respective antigen or receptor, but is not intended to be limited to any one configuration. Antibodies and antibody fragments may be derived from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and any subclass of antibodies (e.g., IgGl, IgG2, IgG3, and IgG4) . The preparation of antibody molecules may be monoclonal or polyclonal. The antibody molecule may also be a human, humanized, CDR-transplanted, or an ex vivo generated antibody. The antibody may have a heavy chain constant region selected from, for example, IgGl, IgG2, IgG3, or IgG4. The antibody may also have a light chain selected, for example, from kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody ".

Antigen-binding fragments of antibody molecules include: (i) Fab fragments as monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) a F (ab ') 2 fragment that is a divalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of the antibody, (v) a diabody (dAb) fragment consisting of the VH domain; (vi) a camelid or camelized variable domain; (vii) single chain Fv (scFv), such as Bird et al. (1988) Science 242: 423-426; And Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883); (viii) Single domain antibody. Such fragments are obtained using conventional techniques known to those skilled in the art and are screened according to the use in the same manner as intact antibodies.

In some embodiments, the antibodies that may be used in the methods and systems described herein may also be single domain antibodies. A single domain antibody may comprise an antibody wherein the complementarity determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, naturally deficient antibodies to light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies that are not antibodies derived from antibodies, and single domain scaffolds . Single domain antibodies may be any single or any future single domain antibodies present in the art. Single domain antibodies can be derived from any species and include, but are not limited to, rats, humans, primates, camels, llamas, fish, sharks, goats, rabbits, and cows. Single domain antibodies are naturally occurring single domain antibodies known as heavy chain antibodies deficient in light chains. Such single domain antibodies are described, for example, in WO 94/04678. For clarity, such variable domains derived from heavy chain antibodies naturally deficient in light chains are known herein as VHH or nanobodies to distinguish them from the conventional VH of the four chain immunoglobulins. These VHH molecule can be derived from the Camelidae (Camelidae) species, for example from an antibody produced in camel, llama, dromedary, alpaca and guanaco. Other species than camelids can naturally produce heavy chain antibodies deficient in light chains; VHH is within the scope of the present invention.

In certain embodiments, the VH and VL regions can be subdivided into hypervariable regions, termed "complementarity determining regions" (CDRs), in which regions of greater conservatism, termed framework regions (FR or FW) have. The range of framework regions and CDRs is precisely defined by several methods (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication 91-3242; Chothia (1987) J. Mol. Biol. 196: 901-917, and the AbM definitions used by Oxford Molecular's AbM antibody modeling software, see, for example, Protein Sequence and Structure Analysis of As used herein, the terms "complementarity determining region ", and" CDR ", " (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, HCDR2, and HCDR3) in each light chain variable region, , LCDR2, LCDR3). The exact amino acid sequence boundaries of the CDRs can be determined using any of a number of well-known methods, such as those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," ("Kabat" numbering scheme), AL-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme) In use, CDRs defined in accordance with the "Chothia" numbering scheme are also often referred to as "hypervariable loops. &Quot;

For example, in the Kabat scheme, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); The CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In the Chothia scheme, the CDR amino acids of VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); The amino acid residues of VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining both the Kabat and the Chothia-type CDR definitions, the CDRs were found to contain amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of human VH and amino acid residues 24-34 LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Unless specifically indicated, the antibody molecule may comprise any combination of one or more Kabat CDRs and / or Chothia hypervariable loops. Under all definitions, each VH and VL comprises three CDRs and four FRs arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An immunoglobulin variable domain sequence refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, the sequence may comprise all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N- or C-terminal amino acids, or may comprise other changes that are compatible with the formation of a protein structure.

In certain embodiments, the antibody generally comprises at least one antigen-binding site or epitope. The term antigen-binding site refers to a portion of an antibody molecule comprising a determinant that forms an interface that binds to a cellular site, or epitope thereof. For proteins (or protein analogs), the antigen-binding site generally includes one or more loops (at least four amino acids or amino acid mimetics) that form an interface that binds to the polypeptide of the cell. Generally, the antigen-binding portion of an antibody molecule comprises at least one or two CDRs and / or hypervariable loops, or more usually at least three, four, five or six CDRs and / or hypervariable loops do.

In some embodiments, different antibodies can compete with each other for binding to a specific epitope or region of a cell. The term " competition "or" cross-compete "is used interchangeably herein to refer to the ability of an antibody molecule to interfere with the binding of another antibody molecule. Interference to binding may be directly or indirectly (e. G., Through antibody molecules or allosteric modulation of the target). Whether antibody molecules can be said to compete with, and thus compete with, the extent to which they can interfere with the binding of another antibody molecule to the target can be determined using competitive binding assays, such as FACS analysis, ELISA or BIACORE analysis Can be determined. In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, the first antibody molecule binds to the target with a second antibody molecule, wherein the binding of the first antibody molecule to the target in the competitive binding assay (e. G., The competition assay described herein) At least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 95% or more, 98% or more, 99% or more. As used herein, the term "epitope" refers to a moiety of an antigen that specifically interacts with an antibody molecule. Such a moiety is referred to herein as an epitope determinant and is generally referred to as including, or being part of, an element such as an amino acid side chain or a side chain. Epitope determinants can be defined by methods known in the art or disclosed herein and can be defined, for example, by crystal analysis or hydrogen-deuterium exchange. At least one or some of the moieties on the antibody molecule that specifically interact with the epitope crystal determinant are generally located in the CDR (s). In general, epitopes have specific three-dimensional structural properties. The epitope may also include specific charge characteristics. While some epitopes are linear epitopes, the other epitopes are stereogenic epitopes.

In some embodiments, the antibody that may be used in the methods described herein may be a monoclonal antibody. As used herein, the term " monoclonal antibody "or" monoclonal antibody composition "refers to a preparation of antibody molecules of a single molecule composition. Monoclonal antibody compositions show one binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced by hybridoma techniques or by methods that do not use hybridoma technology (e. G., Recombinant methods).

In another embodiment, the antibody molecule may be a multiple clone or monoclonal antibody. In other embodiments, the antibody may be produced recombinantly, e. G., By phage display or by a combinatorial method. For example, phage display and combination methods for antibody production are known in the art (see, for example, Ladner et al., US Patent No. 5,223, 409; Kang et al., International Patent Application WO International Patent Application No. WO 92/15679; Breitling et al., International Patent Application No. 92/18619; International patent application WO 92/09690; International patent application WO 90/02809; Fuchs et al. (≪ RTI ID = 0.0 > 1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9 (1991) Nuc Acid Res 19: 4133-4137, and Barbas et al. (1991) PNAS 88: 7978-7982, the entire contents of which are incorporated herein by reference ).

In one embodiment, the antibody is a complete human antibody (e. G., An antibody made in a mouse that has been genetically engineered to produce an antibody from a human immunoglobulin sequence) or a non- human antibody, such as rodent (mouse or rat) , Primates (e.g., monkeys), and camel antibodies. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art. Human monoclonal antibodies can be generated using transgenic mice bearing human immunoglobulin genes rather than mouse systems. Splenocytes from these transgenic mice that have been immunized with the target antigen are used to prepare hybridomas that secrete human monoclonal antibodies (mAbs) having specific affinity for epitopes of human proteins (see, for example, Wood et al. International Application No. WO 91/00906, Kucherlapati et al., PCT Publication No. WO 91/10741, Lonberg et al., International Application No. WO 92/03918, Kay et al., International Application No. 92/03917, Lonberg, N. et al. 1994 Nature 368: 856-859; Green, LL et al 1994 Nature Genet 7: 13-21; Morrison, SL et al 1994 Proc Natl Acad Sci USA 81: 6851-6855 Bruggeman et al. 1993 Year Immunol 7: 33-40; Tuaillon et al 1993 PNAS 90: 3720-3724; Bruggeman et al 1991 Eur J Immunol 21: 1323-1326). The antibody may be a variable region or a portion thereof, e.g., a CDR is produced in a non-human organism, such as a rat or a mouse. Chimeric, CDR-transfected, and humanized antibodies can be used. Antibodies are generated in non-human organisms, such as rats or mice, and then modified, e.g., in a variable framework or constant region, to reduce antigenicity in humans. Chimeric antibodies can be prepared by recombinant DNA techniques known in the art (Robinson et al., International Patent Application No. PCT / US86 / 02269; Akira, et al., European Patent Application 184,187; Taniguchi, Cabilly et al., US Patent 4,816, 567; Cabilly et al., European Patent Application 125, 023; Better et al < RTI ID = 0.0 > (1987) PNAS 84: 3439-3443; Liu et al., 1987, J. Immunol. 139: 3521-3526; Sun et al. (1985) Nature 314: 446-449, and Shaw et al., 1988, J. Natl Cancer Inst. ≪ RTI ID = 0.0 > 80: 1553-1559).

In certain embodiments, a humanized or CDR-grafted antibody will have at least one or two, but generally all three, receptor CDRs (in the immunoglobulin heavy chain and light chain) replaced with a donor CDR. Antibody non-human CDRs, or only a portion of the CDRs can be replaced with non-human CDRs. It may be desirable to replace the number of CDRs required for binding of humanized antibodies to a particular epitope. Preferably, the donor will be a rodent antibody, such as a rat or mouse antibody, and the receptor will be a human framework or a human consensus framework. Generally, an immunoglobulin that provides a CDR is termed a "donor ", and an immunoglobulin that provides a framework is termed a" receptor ". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The receptor framework is a consensus framework, or sequence, of a naturally occurring (e.g., human) framework or at least about 85%, preferably 90%, 95%, 99% As used herein, the term "consensus sequence " refers to a sequence formed from the most frequently occurring amino acid (or nucleotide) in the related sequence class (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987. In the protein class, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the class. If two amino acids occur equally frequently, either consensus sequence A "consensus framework" refers to a framework region within a consensus immunoglobulin sequence.

In some embodiments, the antibody can be humanized by methods known in the art (see, for example, Morrison, S. L., 1985, Science 229: 1202-1207, Oi et al, 1986, BioTechniques 4: 214, et al., U.S. Patent No. 5,585,089, U.S. Patent No. 5,693,761 and U.S. Patent No. 5,693,762, the contents of which are incorporated herein by reference). Humanized or CDR-grafted antibodies can be generated by CDR-grafting or CDR substitution, wherein one, two or all CDRs of the immunoglobulin chain can be substituted. See, for example, U.S. Patent Nos. 5,225,539; Jones et al. 1986 Nature 321: 552-525; Verhoeyan et al. 1988 Science 239: 1534; Beidler et al. 1988 J. Immunol. 141: 4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of which are expressly incorporated herein by reference. Winter describes a CDR-grafting method that can be used to produce humanized antibodies (British Patent Application GB 2188638A, Mar. 26, 1987; Winter, US Patent No. 5,225,539), the contents of which are incorporated herein by reference Are explicitly included. Humanized antibodies in which a specific amino acid has been substituted, removed or added can also be used. Standards for amino acid selection from donors are described, for example, in U.S. Patent No. 5,585,089, for example, in paragraphs 12-16 of U.S. Patent No. 5,585,089, for example, in U.S. Patent No. 5,585,089, paragraphs 12-16, Are incorporated herein by reference. Other techniques for humanizing antibodies are described in Padlan et < RTI ID = 0.0 > al., ≪ EP 519596 A1.

In some embodiments, the antibody molecule may be a single chain antibody. Single-chain antibodies (scFV) can be engineered (see, for example, Colcher, D. et al. (1999) Ann NY Acad Sci 880: 263-80; and Reiter, Y. (1996) Clin Cancer Res 2: 245-52). Single chain antibodies can be dimerized and multimerized to produce multivalent antibodies with specificity for different epitopes of the same target protein. In another embodiment, the antibody molecule is selected from heavy chain constant regions of, for example, IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; Specifically, it has a heavy chain constant region selected from, for example, IgGl, IgG2, IgG3, and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, kappa or lambda (e.g., human) light chain constant region. The constant region may be modified to alter the properties of the antibody (e.g., to increase or decrease one or more of the following properties: Fc receptor binding, antibody glucosylation, number of cysteine residues, effector cell function, and / or complement function) , E. G., Mutated. In one embodiment, the antibody has an effector function; Complement can be complemented. In other embodiments, the antibody does not utilize an effector cell; Or complement. In another embodiment, the antibody has reduced ability or no ability to bind to the Fc receptor. For example, an isotype or subtype, fragment or other mutation that does not support binding to an Fc receptor, such as a mutated or deleted Fc receptor binding region.

Methods for altering antibody constant regions are known in the art. An antibody with altered function, such as an altered affinity for an effector ligand, such as an FcR on a cell, or a C1 component of a complement, can be produced by replacing at least one amino acid residue in the constant region of the antibody with a different residue (see, e.g., EP 388,151 A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260, the contents of which are incorporated herein by reference in their entirety). Similar types of modifications that can reduce or eliminate this function can be described when applied to murine or other species of immunoglobulin.

In certain embodiments, the antibody may be derivatized or conjugated to another functional molecule (e.g., another peptide or protein), or conjugated to a label. As used herein, a "derivatized" antibody is a modified antibody. Derivatization methods include, but are not limited to, addition of fluorescent moieties, light scattering moieties, and luminescent moieties that are complexed with affinity ligands such as metals, radioactive nucleotides, toxins, enzymes or biotin. Antibodies may include antibodies in derivatized and otherwise modified forms, including immunomodulatory molecules. For example, an antibody molecule can be conjugated to one or more other molecular entities such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, one or more labels, a cytotoxic agent, a drug, and / (Such as a chemical bond, a genetic fusion, a noncovalent bond, or otherwise) to mediate binding of another molecule to an antibody or antibody portion (e.g., a streptavidin core region or a polyhistidine tag). One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (to produce the same type or different types of, e.g., bispecific antibodies). Suitable cross-linking agents include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., homobifunctional) monomers having two different reactive groups separated by a suitable spacer ) (Such as di-succinimidylsuberate) crosslinking agents. Such linkers are available from Pierce Chemical Company, Rockford, Ill.

In some embodiments, the antibody may comprise a detectable label that is selected based on the specific technique performed in the cell analyzer. Examples of useful detectable agents that can be derivatized (or labeled) with the antibody molecule include fluorescent compounds, various enzymes, complementary molecules, luminescent materials, bioluminescent materials, fluorescent luminescent metal atoms such as europium (Eu) And phosphorescent metal atoms, and transition metals, lanthanide elements, and radioactive materials (described below). Exemplary fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, picoerythrine, and the like. Antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, mustard peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase, and the like. When an antibody is derivatized to a detectable enzyme, the enzyme is detected by adding an additional reagent that is used to produce a detectable reaction product. For example, when a detectable substance, mustard peroxidase, is present, the addition of hydrogen peroxide and diaminobenzidine induces a colored reaction product, which is detectable. Antibody molecules may also be derivatized with complementary molecular moieties (e.g., streptavidin / biotin and avidin / biotin). For example, antibodies can be derivatized to biotin and detected through indirect measurement of avidin or streptavidin binding. Other examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dysyl chloride or fcoerythrin; Examples of luminescent materials include luminol, examples of bioluminescent materials include luciferase, luciferin, and aquoline.

Labeled antibody molecules can be used to characterize one or more phenotypes or biological responses of a cell, for example, using a cell analyzer. Antibodies are generally used to assess the abundance and pattern of protein expression and to monitor protein levels in tissues as part of a clinical testing procedure, for example, to determine the efficacy of a given therapeutic regimen, To differentiate a particular cell, it is used to detect the presence (or absence) of the antigen.

In certain embodiments, the antibody molecule may be conjugated to another molecular entity, generally a marker or therapeutic agent (e. G., A cytotoxic or cellular proliferation inhibitor) or moiety. Radioactive isotopes can be used in diagnostic or therapeutic applications and can also be determined using the mass spectrometry methods and systems described herein. Radioactive isotopes that can be conjugated to the antibody include, but are not limited to, alpha-, beta-, or gamma-emitters, or beta- and gamma-emitters. These radioactive isotopes of iodine ⁽¹³¹ I or ¹²⁵ I), yttrium ⁽⁹⁰ Y), lutetium ⁽¹⁷⁷ Lu), actinium ⁽²²⁵ Ac), pre-aged audio di, astatine ⁽²¹¹ At), rhenium ⁽¹⁸⁶ Re ), bismuth ⁽²¹² Bi or ²¹³ Bi), indium ⁽¹¹¹ in), technetium ^(99m Tc), the ⁽³² P), rhodium ⁽¹⁸⁸ Rh), sulfur ⁽³⁵ S), carbon ⁽¹⁴ C), tritium ⁽³ H), comprises a chromium ⁽⁵¹ Cr), chlorine ⁽³⁶ Cl), cobalt ⁽⁵⁷ Co or ⁵⁸ Co), iron ⁽⁵⁹ Fe), selenium ⁽⁷⁵ Se), or gallium ⁽⁶⁷ Ga), but are not limited to . Useful radioactive isotope as a therapeutic agent, yttrium ⁽⁹⁰ Y), lutetium ⁽¹⁷⁷ Lu), actinium ⁽²²⁵ Ac), pre-aged audio di, astatine ⁽²¹¹ At), rhenium ⁽¹⁸⁶ Re), bismuth ⁽²¹² Bi or ²¹³ Bi), and rhodium ( ¹⁸⁸ Rh). As for example, a useful radioactive isotopes for use in the diagnosis as a cover, iodine ⁽¹³¹ I or ¹²⁵ I), indium ⁽¹¹¹ In), technetium (99 ^m Tc), the ⁽³² P), carbon ⁽¹⁴ C), And tritium ( ³ H), or one or more of the above listed therapeutic isotopes. The mass spectrometer can be used to detect whether these radioisotopes have been absorbed by a particular cell and how much radioisotope is present in each individual cell.

As discussed above, antibody molecules can also be conjugated to therapeutic agents. Therapeutically active radioisotopes have already been mentioned. Examples of other therapeutic agents include Taxol, cytokalacin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, Dihydroidostosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoid such as meiocanthoid, (See U.S. Patent No. 5,208,020), CC-1065 (see U.S. Patent Nos. 5,475,092, 5,585,499, 5,846, 545), and analogues or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decabazine), alkylating agents (such as mechlorethamine, thiotepachlorambucil, CC-1065, melphalan, carmustine (BSNU) and rosuvastatin (CCNU), cyclosporpha mide, pilaster, dibromomonitol, stetozotocin, mitomycin C, and cis-dichlorodiamine platinum (DDP) x splatin), anthracyclines (e.g., daunorubicin (past, daunomycin) and doxorubicin), antibiotics (such as dactinomycin (past actinomycin), bleomycin, Anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, tosol and maytansinoid). If desired, the antibody may be conjugated to both the therapeutic agent and the detectable label. The efficacy of treatment using therapeutic conjugated antibodies can be assessed using the systems and methods described herein.

In certain embodiments, the antibody molecule is a multispecific (e. G., Bispecific or triple-specific) antibody molecule. Protocols for producing bispecific or heterodimeric antibody molecules are known in the art and include, for example, but are not limited to the following: "knob in hole" 5,731,168; Electrostatic steering Fc pairing, for example as described in WO 09/089004, WO 06/106905 and WO 2010/129304; Strand exchange operative domain (SEED) heterodimer formation, for example as described in WO 07/110205; Fab arm exchange, for example as described in WO 08/119353, WO 2011/131746, and WO 2013/060867; Dual antibody conjugates by antibody bridging to produce bispecific structures using heterobifunctional reagents having amine-reactive groups and sulfhydryl-reactive groups, for example as described in U.S. Patent No. 4,433,059; Bispecific antibody determinants generated by recombination of half-antibodies (heavy chain-light chain pairs or Fabs) from different antibodies via reduction and oxidation cycles of two heavy chain disulfide bonds, for example as described in U.S. Patent No. 4,444,878; Trifunctional antibody, e. G., Three Fab 'fragments bridged through a sulfhydryl-reactive group, such as described in U.S. Patent No. 5,273,743; Biosynthetic binding proteins such as pairs of scFvs bridged through the C-terminal tail, preferably via disulfide or amine-reactive chemical cross-linking, as described, for example, in U.S. Patent No. 5,534,254; Fab fragments having different binding specificities dimerized via antibodies of dual functionality, e. G., Leucine zippers (e. G., C-fos and c-jun) replacing the constant domains, such as those described in U.S. Patent No. 5,582,996; (2 Fab fragments) linked via a polypeptide spacer between the CH1 region of one antibody and the VH region of another antibody having a light chain generally associated with a polypeptide (such as the VH-CH1 region of the two antibodies) oligospecific single- and oligovalent receptors such as those described in U.S. Patent No. 5,591,828; Bispecific DNA-antibody conjugates, such as cross-linking antibodies or Fab fragments through double-stranded fragments of DNA, see, for example, U.S. Patent No. 5,635,602; A bispecific fusion protein, such as an expression construct containing a scFv having a hydrophilic helical peptide linker between, for example, two scFvs and a fully constant region, such as described in U.S. Patent No. 5,637,481; Multivalent and multispecific binding proteins, such as a dimer of a polypeptide having a first domain having a binding region of an Ig heavy chain variable region and a second domain having a binding region of an Ig light chain variable region, (Higher dimensional structures are also included in bispecific, triple specific, or quad-specific molecule production, for example as described in U.S. Patent No. 5,837,242; linked VLs further connected to the peptide spacer in the antibody hinge region and CH3 region And VH chains, which can be dimerized to form bispecific / polyvalent molecules, such as those described in U.S. Patent No. 5,837,821; short peptide linkers (e. G., 5 or 10 amino acids) VH and VL domains that do not have a linker in the direction, dimers to form bispecific diabodies, (See, for example, U.S. Patent No. 5,844,094), a VH domain linked by a peptide bond to a cross-linking group at the C-terminus, which is further bound to the VL domain to form a series of FV (or scFv) Or a family of VL domains within a family member, such as those set forth in U.S. Patent No. 5,864,019; and single chain binding polypeptides having both VH and VL domains linked through a peptide linker can be linked to a multivalent structure via noncovalent or chemical cross-linking Linked using scFV or diabody type formats to form, for example, homologous, heterologous, tridentate and tetrahedral structures, such as, for example, U.S. Patent 5,869,620. Additional exemplary multispecific and bispecific Molecules and methods for their preparation are found in the following references: for example, U.S. Patent No. 5,910,573, U.S. Patent No. 5,932,448, U.S. Patent No. 5,959,083 , U.S. Patent No. 5,989,830, U.S. Patent No. 6,005,079, U.S. Patent No. 6,239,259, U.S. Patent No. 6,294,353, U.S. Patent No. 6,333,396, U.S. Patent No. 6,476,198, U.S. Patent No. 6,511,663, U.S. Patent No. 6,670,453, U.S. Patent Nos. 6,743,896, 6,809,185, 6,833,441, 7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866, 7,612,181 Lake, US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1, US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1, US2007004909A1, US2007087381A1, US2007128150A1 , US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1, US20081 52645A1, US2008171855A1, US2008241884A1, US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1, US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2, WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2, WO9964460A1.

In another embodiment, an antibody molecule (e.g., a monospecific, bispecific, or multispecific antibody molecule) is conjugated to another partner, such as a protein, e.g., one or more cytokines, Covalently bonded, e. G., As a fusion protein. In other embodiments, the fusion molecule comprises one or more proteins, for example, one or more cytokines. In one embodiment, the cytokine is an interleukin (IL) selected from one, two, three or more of IL-1, IL-2, IL-12, IL-15 or IL-21. In one embodiment, the bispecific antibody molecule has a first binding specificity on a first target, a second binding specificity on a second target, and an interleukin (e.g., IL-12) Lt; / RTI > "Fusion protein" and "fusion polypeptide" refer to polypeptides that have at least two portions covalently linked together, each of which is a polypeptide having a different property. The properties may be biological properties such as in vitro or in vivo activity. Such properties may also be simple chemical or physical properties such as binding to the target molecule, reaction catalysis, and the like. The two moieties may be linked directly through a single peptide bond or peptide linker, but are within one another in the reading frame. If desired, the fusion protein may comprise a detectable label detectable using a cell analyzer as described herein.

In the example where the magnetic label is used with a magnetic cell sorter, the magnetic label may comprise magnetic, semi-magnetic or super-magnet nanoparticles with an average size of generally about 50-100 nm. Self-labeling can be placed inside the column so that cells that pass through the column and specifically bind to the self-label are captured. Cells that do not bind to self-labels can pass. The bound cells can be eluted later by turning off the magnetic field and allowing the cells to elute from the column. In some embodiments, Dynabeads or other spherical beads of larger size, e.g., 1-5 microns on average, may be used instead. Suitable self-labels and beads are available, for example, from Miltenyi Biotec Inc. (Auburn, Calif.).

Specific embodiments are set forth in order to provide a better understanding of some of the novel features disclosed herein.

Example 1

One example of a system that can be used to measure one or more characteristics of an individual cell and to measure the amount of at least one element of an individual cell is shown in FIG. The system 1100 includes a syringe 1110, a loop 1120, a microtiter plate 1130, a valve / switch 1140, and a flow cell analyzer 1150 fluidly coupled to the mass spectrometer (not shown) do. Loop 1120 may be configured to maintain a volume of about 100-300 microliters. Loop 1120 may be used to provide a linear flow into flow cytometer 1150. [ In use of system 1100, loop 1120 may be filled by syringe 1110. The valve / switch 1140 may be operated to cause cells to be provided from the microtiter plate 1130 to the flow cytometer 1150. As each individual cell passes through the flow cytometer 1150, a pulse in the flow cytometer 1150 can be correlated with a pulse in a mass spectrometer (not shown). Different measurements can be correlated to provide information about the cell's metal content and biological response.

Example 2

Referring to Fig. 12, there is shown a graph showing possible data that can be acquired and used to correlate the measured metal mass and cell size of each cell. As can be seen in Fig. 12, the intracellular metal content increases substantially linearly with increasing cell size. Such data may indicate a linear uptake of the metal agonist into the cell depending on the cell size.

Example 3

Referring to FIG. 12, another graph is shown showing possible data that can be acquired and used to correlate the measured metal mass and cell size of each cell. As can be seen in Figure 13, there is a cluster of metal levels as a function of cell size as the cell size increases.

Example 4

Referring to Figures 14A and 14B, the graphs show viable cell or apoptotic cell to metal content. In Fig. 14A, the living cell 1410 has a higher content of metal. 14B shows that apoptotic cells 1420 have a lower metal content than the live cells 1410. FIG. Whether or not each cell is alive or dead can be determined using a flow cytometer or other cell analyzer and the metal content of the particular cell can be determined using a mass spectrometer.

Example 5

Referring to FIG. 15, there is shown a graph in which four different fluorescent markers (shown in different shapes shown) are shown for metal content. Each of the different fluorescent markers can be, for example, on different antibody molecules. Flow cytometry analyzers can be used to measure the presence of fluorescent labels as an indicator of antibody binding. Individual cells can then be provided to a mass spectrometer to determine the amount of intracellular metals. The data may be correlated as shown in FIG.

When introducing elements of the embodiments disclosed herein, the terms "a", "an", "the" and "the" are intended to mean that there is more than one element. The terms "comprising," "including" and "having" are intended to be open-ended and that there may be additional elements other than the other listed elements. It will be apparent to those skilled in the art, in view of the benefits of the present invention, that the various components of the embodiments may be interchanged or replaced with various components in other embodiments.

While specific embodiments, configurations, embodiments and embodiments have been described above, those skilled in the art will recognize that additions, substitutions, changes, and variations of the disclosed exemplary aspects, configurations, embodiments, and embodiments are possible in light of the benefit of the present disclosure .

Claims (45)

A method comprising the steps of:
Providing a mass spectrometer from an individual cell cell analyzer within the cell population to quantify the amount of at least a first element in the provided individual cells; And
Correlating the quantified amount of the first element in the provided individual cells with the measurement of the individual cells from the cell analyzer. 2. The method of claim 1, further comprising quantifying the amount of the first element in each cell of the cell population using a mass spectrometer, and correlating the quantified amount of each first element of the cell with individual cell measurements from the cell analyzer Methods of inclusion. 3. The method of claim 2, further comprising determining the number of cells in the cell population comprising the first element, using the quantified amount of the first element of each cell and the individual cell measurements from the cell analyzer. 3. The method of claim 2, further comprising determining the number of cells in the population of cells exhibiting the selected biological response, using the quantified amount of the first element of each of the cells and individual cell measurements from the cell analyzer. 3. The method of claim 2, further comprising correlating the quantified amount of the first element with the cell size using the quantified amount of the first element of each cell and the individual cell measurements from the cell analyzer. 3. The method of claim 2, further comprising quantifying at least a second element in each cell of the cell population. 3. The method of claim 1, further comprising the step of providing a single cell suspension to a cell analyzer after constructing a population of cells with the single cell suspension. 8. The method of claim 7, further comprising constructing a cell analyzer to provide single cell eukaryotic cells to a mass spectrometer or single cell prokaryotic cells to a mass spectrometer. 9. The method of claim 8, further comprising configuring the cell analyzer as a flow cytometer. 9. The method of claim 8, further comprising configuring the cell analyzer as a fluorescence activated cell sorter. 9. The method of claim 8, further comprising configuring the cell analyzer as a magnetic classifier. The method of any one of claims 9-11, further comprising configuring the mass spectrometer to include an inductively coupled plasma. 13. The method of claim 12, further comprising the step of separating the cell population from other species using chromatography and then providing the cell population to a cell analyzer. 13. The method of claim 12, further comprising determining cell size as a measure of individual cells from the cell analyzer. 13. The method of claim 12, further comprising determining cell viability as a measure of individual cells from the cell analyzer. 13. The method of claim 12, further comprising determining cell phenotype using a specific label as a measure of individual cells from a cell analyzer. 13. The method of claim 12, further comprising determining cell health using a specific label as a measure of individual cells from the cell analyzer. The method of claim 1, further comprising the step of constructing the cell such that the population of cells comprises an average size of between 0.2 microns and 100 microns, constituting the cell analyzer as a flow cytometer and constructing the mass spectrometer to include an inductively coupled plasma How to. The method of claim 1 further comprising the steps of separating the organelles from the cell population, providing the individual organelles from the separated organelles to the mass spectrometer from a cell analyzer to quantify the amount of the first element in the provided individual organelles, Further comprising the step of correlating the quantified amount of element with a measure of individual organotypes from a cell analyzer. The method of claim 1, further comprising correlating the quantified amount of at least a first element in an individual cell provided using the processor with a measure of an individual cell from the cell analyzer. A method for measuring the uptake of a first metal agonist into a cell, comprising the steps of:
Exposing the population of cells to a first metal agonist;
Determining the phenotype of individual cells in the cell population using a cell analyzer;
Providing individual cells from the cell analyzer to a fluid coupled mass spectrometer in a cell analyzer to quantify the amount of the second metal agent absorbed into each of the provided individual cells; And
Correlating the quantified amount of the second metal agent absorbed into each of the individual cells with the determined phenotype of each of the individual cells. 22. The method of claim 21, further comprising configuring the mass spectrometer to include an inductively coupled plasma. 22. The method of claim 21, further comprising selecting a first metal agent as a transition metal agent or metal agent comprising an atomic weight of from 2 to 285 amu of an element. 24. The method of claim 23, further comprising the step of selecting the transition metal agonist as a DNA replication inhibitor and selecting the cell population as a human cell. 25. The method of claim 24, further comprising quantifying the amount of cells in the population of cells that are resistant to the absorption of the transition metal agonist using the quantified amount of individual intracellular metal agonists and the determined phenotype of each individual cell in the population of cells How to. 22. The method of claim 21, further comprising the step of exposing the cell to a second metal agonist, wherein the second metal agonist comprises a metal different from the first metal agonist, the cell population exposed to the second metal agonist using a cell analyzer Determining the phenotype of the individual cells, providing the individual cells from the cell analyzer to a fluid-coupled mass spectrometer in a cell analyzer to quantify the amount of the second metal agent absorbed into each of the provided individual cells, And correlating the quantified amount of the second metal agent to the determined phenotype of each of the individual cells. 27. The method of claim 26, further comprising quantifying the amount of cells in the population of cells that are resistant to the uptake of the second metal agonist using the quantified amount of the second metal agent in the individual cell and the determined phenotype of each of the individual cells of the population of cells ≪ / RTI > 22. The method of claim 21, further comprising constructing a cell analyzer as one of a flow cytometer, a fluorescence activated cell sorter, and a magnetic classifier. 22. The method of claim 21, further comprising determining at least one of cell size or cell viability as a determined phenotype of the cell. 22. The method of claim 21, further comprising the step of marking the cell with a specific label to determine the phenotype of the cell. A system configured to correlate a cell phenotype with a quantity of a first element of a cell, comprising:
A cell analyzer configured to provide individual cells from the cell population and determine cell measurements of the provided individual cells;
A mass spectrometer coupled to the cell analyzer and adapted to receive individual cells provided from the cell analyzer, the mass spectrometer being configured to determine the amount of the first element present in the provided individual cells; And
And correlate the determined cell measurements of the provided individual cells with the amount of the first element present in the provided individual cells. 32. The system of claim 31, wherein the cell analyzer is configured as a flow cytometer. 33. The system of claim 32, wherein the mass spectrometer comprises an inductively coupled plasma. 34. The system of claim 33, further comprising a cell-introducing device fluidly coupled to the flow cytometer, wherein the cell-introducing device comprises a loop configured to provide the cells in a linear flow to the flow cytometer. 35. The system of claim 34, wherein the cell introduction device further comprises a syringe. 35. The system of claim 34, wherein the cell introduction device comprises an autosampler. 32. The system of claim 31, wherein the cell analyzer is configured as a fluorescence activated cell sorter. 32. The system of claim 31, wherein the cell analyzer is configured as a magnetic classifier. 32. The system of claim 31, comprising a sample introduction device fluidly coupled to each of a cell analyzer and a mass spectrometer, wherein the sample introduction device is configured to receive individual cells from the cell analyzer and to provide the individual cells received to the mass spectrometer. 41. The system of claim 39, wherein the cell introduction device comprises a spray chamber. 41. The system of claim 40, wherein the mass spectrometer is configured to determine an amount of the first metal as the first element. 41. The system of claim 40, wherein the mass spectrometer comprises an ion source fluidly coupled to the spray chamber, a mass analyzer fluidly coupled to the ion source, and a detector fluidly coupled to the mass analyzer. 41. The system of claim 40, wherein the cell analyzer is configured to determine a cell size, wherein the processor is configured to correlate the determined cell size of the individual cells with a determined amount of the first element present in the provided individual cells. 41. The system of claim 40, wherein the cell analyzer is configured to determine cell viability and wherein the processor is configured to correlate the determined cell viability of the individual cells with the determined amount of the first element present in the provided individual cells. 41. The system of claim 40, wherein the cell analyzer is configured to determine a cell phenotype, wherein the processor is configured to correlate the determined cell phenotype of the individual cell with a determined amount of the first element present in the provided individual cell.

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