CN110608991A - Cell cycle detection kit based on mass flow detection technology and detection method - Google Patents

Cell cycle detection kit based on mass flow detection technology and detection method Download PDF

Info

Publication number
CN110608991A
CN110608991A CN201910846725.5A CN201910846725A CN110608991A CN 110608991 A CN110608991 A CN 110608991A CN 201910846725 A CN201910846725 A CN 201910846725A CN 110608991 A CN110608991 A CN 110608991A
Authority
CN
China
Prior art keywords
solution
volume
cell
liquid
parts
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910846725.5A
Other languages
Chinese (zh)
Other versions
CN110608991B (en
Inventor
王平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Universal Health Technology Co Ltd
Original Assignee
Zhejiang Universal Health Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Universal Health Technology Co Ltd filed Critical Zhejiang Universal Health Technology Co Ltd
Priority to CN201910846725.5A priority Critical patent/CN110608991B/en
Publication of CN110608991A publication Critical patent/CN110608991A/en
Application granted granted Critical
Publication of CN110608991B publication Critical patent/CN110608991B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Dispersion Chemistry (AREA)
  • Toxicology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biophysics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a cell cycle distribution kit based on a mass flow detection technology and a detection method thereof, which utilize the mass flow detection technology to determine the proportion of cell cycles of cells of each subgroup in a sample at a single cell level aiming at a small amount of cells of clinical and scientific research tissue samples, thereby revealing the influence of the change of the proliferation capacity of the specific cell subgroup in the sample on the physiological and pathological states of the sample.

Description

Cell cycle detection kit based on mass flow detection technology and detection method
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of cell biology, in particular to a cell cycle detection kit and a cell cycle detection method based on a mass flow detection technology.
[ background of the invention ]
The mass flow detection technology is a technology for detecting rare metal labeled antibodies through a mass spectrometer on the single cell level and performing multi-parameter and high-flux qualitative analysis on each cell. Compared with the traditional flow cytometry, the technology has the advantages of more detection parameters and higher signal-to-noise ratio, is the most advanced single-cell flow detection technology at present, and is more and more widely applied in the fields of clinical medicine and biological research.
The cell cycle refers to the overall course of changes that a cell undergoes from the end of the last cell division to the end of the next cell division. The process of cell division can be divided into two stages, inter-division and division, depending on whether the process of cell division occurs or not. Furthermore, the interphase can be further divided into a DNA synthesis early stage (G1 stage), a DNA synthesis late stage (G2 stage) and a DNA synthesis late stage (S stage) according to the replication of replication-related proteins, enzyme molecules and genetic materials, and then the mother cell distributes the genetic materials, organelles and nutrients, etc. into two daughter cells uniformly to complete the division stage (M stage). In addition, the proliferating cells may also be regulated to temporarily escape the cell cycle to stop cell division, referred to as the G0 phase. Thus, cells in different cell cycle states of G0, G1, S, G2, M phases collectively characterize the proliferative state of the cell population.
The cell cycle is used as a cell biological parameter for indicating the proliferation state of a cell population, and has important research significance for detecting cells of different tissue types under physiological or pathological states. The tissues and organs of the body maintain normal physiological functions, cellular metabolism and damage repair functions. The cells of the tissue and organ in the cell cycle produce daughter cells which are of great significance for supplementing the cells with damaged, aged or dead functions in the tissue and organ. In addition, if the cell cycle regulation of cells in the tissue organ is abnormal, the cell proliferation rate is accelerated, the physiological function disorder of the tissue organ can be caused, and the function change of the tissue organ related to the tumor can be caused. However, a tissue organ is a group of cells formed by orderly cooperation and organic constitution of a plurality of types of cells, and has high heterogeneity. The cell cycle of cells measured at the global tissue level does not specifically reflect the proliferation status of different types of cells in the tissue organ. Therefore, the method for detecting the cell cycle state of each cell in the tissue and organ from the single cell level has important research and clinical diagnosis significance for revealing the influence of the proliferation activity of various tissue cells on the physiological functions of the tissue and organ.
The cell cycle detection means commonly used in scientific research at present still mainly uses the flow cytometry technology for detection, a target cell subset to be detected in a sample is separated and selected through specific and limited extracellular antibody staining, and then cell cycle analysis is carried out by using PI staining or a cell cycle detection kit provided by other companies. At present, the main commercial cell detection kit cannot well distinguish cells in M phase and G0 phase, and has the defects of long sample cell processing period (sorting is needed before detection), low sample detection flux (only one sample can be detected each time), poor detection result stability (batch effect possibly existing due to sample cell state change and asynchronous processing and detection caused by overlong sample living cell processing time) and the like. Therefore, currently commercially available cell cycle detection technologies cannot evaluate the cell cycle states of different types of tissue cells existing in the same sample from a single cell level, can only be characterized from a cell population level, and research means cannot meet the requirement of describing the cell proliferation states of different types of cells in a sample tissue with low sample volume and high throughput.
[ summary of the invention ]
The invention aims to provide a cell cycle detection kit based on a mass flow detection technology, which can detect the cell cycle state of sample cells on a single cell level and can meet the requirements of low sample volume and high throughput in commercial application.
The invention also aims to provide a cell cycle detection method based on the mass spectrometry flow detection technology by using the kit.
The technical solution of the invention is as follows:
the cell cycle detection kit based on the mass flow detection technology is characterized by comprising LD liquid, I liquid, FI liquid, B liquid, W liquid, P liquid, D liquid and BC1-BC10 liquid;
the LD liquid comprises 194Pt and RPMI1640 basic culture medium;
the liquid I comprises iodoglycoside and dimethyl sulfoxide (DMSO);
the F I liquid comprises a fixed liquid and a permeable liquid;
the F II solution comprises formaldehyde and a phosphate buffer solution;
the solution B is a blocking buffer solution which contains human immunoglobulin, mouse immunoglobulin, rat immunoglobulin, hamster immunoglobulin, bovine serum albumin and phosphate buffer solution and is used for flow cytometry;
the W liquid is a transparent buffer liquid;
the liquid P is a fixed/permeable liquid;
the solution C is a cell cycle antibody staining solution;
the solution D comprises 191/193Ir, formaldehyde and phosphate buffer solution;
the BC1-BC10 liquid is a liquid comprising phosphate buffer and any two of Pd104, Pd105, Pd106, Pd108 and Pd 110.
Further, the LD solution was 1 part by volume of RPMI1640 basic medium and 0.01 part by volume of 1mM194Pt (201194, Fluidigm). The explanations in parentheses refer to the product number and the manufacturer, both of which are commercially available products, as follows.
Further, the solution I comprises 0.354 to 17.705 parts by mass of iodoglycoside and 1 part by volume of dimethyl sulfoxide (Sigma), wherein the parts by mass are in mg units and the parts by volume are in mL units.
Further, the above-mentioned F I liquid was 5 Ximmobilized and liquid-permeable (Fluidigm).
Further, the FII solution is 0.9-1.3 parts by volume of a 16 wt% formaldehyde solution and 9 parts by volume of a phosphate buffer solution (GNM20012), wherein the parts by volume are measured in mL.
Further, the solution B is 0.5 to 2 parts by volume of a human immunoglobulin solution having a concentration of 2mg/mL, 0.5 to 2 parts by volume of a mouse immunoglobulin solution having a concentration of 2mg/mL, 0.5 to 2 parts by volume of a rat immunoglobulin solution having a concentration of 2mg/mL, 0.5 to 2 parts by volume of a hamster immunoglobulin solution having a concentration of 2mg/mL, 0.5 to 2 parts by mass of bovine serum albumin (mass to volume ratio, compared to the total volume of the solution B), and 100 parts by volume of a phosphate buffer (GNM20012), wherein the parts by mass are in mg and the parts by volume are in mL.
Further, the W solution was 1 part by volume of 10 × permeation solution (Invitrogen) and 9 parts by volume of water, wherein the parts by volume were measured in mL.
Further, the liquid P was 1 part by volume of a fixed/permeation concentrated solution (Invitrogen) and 3 parts by volume of a fixed/permeation diluted solution (Invitrogen), wherein the parts by volume were measured in mL.
The solution C was a cell cycle antibody staining solution prepared from 1 part by volume of CyclinB1-153Eu (1:100, Fluidigm), 1 part by volume of phospho-Histone H3-165Ho (1:100, Biolegend), 1 part by volume of phospho-Rb-150Nd (1:100, Fluidigm), 0.5-2 parts by mass of bovine serum albumin, and 97 parts by volume of phosphate buffer (GNM 20012). Wherein, the mass part is mg as a measurement unit, and the volume part is mL as a measurement unit. And the metal channel marked by each antibody in the solution C can be prepared according to the requirements of customer projects.
Further, the D liquid was 0.0005 part by volume of Cell-IDTMIntercalator-ir (fluidigm), 0.09-0.13 parts by volume of a 16 wt% formaldehyde solution, 0.9 parts by volume of a phosphate buffer (GNM20012), wherein parts by volume are measured in mL.
Further, the BC (1-10) solution was:
BC1 solution contains 1 part by volume of Pd104 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC2 solution contains 1 volume part of Pd106 (1.5-2. mu.M, Fluidigm), 1 volume part of Pd108 (1.5-2. mu.M, Fluidigm), 100 volume parts of phosphate buffer (GNM 20012);
BC3 solution contains 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC4 solution contains 1 part by volume of Pd104 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd106 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC5 solution contains 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd108 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC6 solution contains 1 part by volume of Pd106 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC7 solution contains 1 volume part of Pd104 (1.5-2. mu.M, Fluidigm), 1 volume part of Pd108 (1.5-2. mu.M, Fluidigm), 100 volume parts of phosphate buffer (GNM 20012);
BC8 solution contains 1 part by volume of Pd105 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd106 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC9 solution contains 1 part by volume of Pd104 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), 100 parts by volume of phosphate buffer (GNM 20012);
BC10 solution contained 1 part by volume of Pd108 (1.5-2. mu.M, Fluidigm), 1 part by volume of Pd110 (1.5-2. mu.M, Fluidigm), and 100 parts by volume of phosphate buffer (GNM 20012).
Wherein, each sample to be detected is dyed by selecting one of BC1-BC10 liquid, different BC liquid is required to be selected for different samples, and at most 10 different samples to be detected can be detected simultaneously. Parts by volume are measured in mL.
Further, LD solution, I solution, FI solution, B solution, W solution, P solution, C solution, D solution, and BC1-BC10 solution were prepared on ice and stored at 2-8 ℃.
The cell cycle detection method based on the mass flow detection technology is characterized in that the kit is used, and comprises the following steps:
step one, preparing a sample single cell suspension: collecting single cell suspension, each sample containing 1-5 × 106The cell suspension of individual cell amount was centrifuged at 400g at room temperature for 5min, and the supernatant was carefully discarded to obtain a cell pellet.
Step two, determining cell death and cell viability: 1mL of LD solution was added, and the cell pellet was gently aspirated by a pipette to fully resuspend the pellet, followed by incubation in a cell incubator at 37 ℃ for 5 min. After addition of 5mL of serum-free RPMI1640 medium, the mixture was centrifuged at 400g for 5min at room temperature, and the supernatant was carefully discarded to obtain a cell pellet.
Step three, IdU incubation: and (3) adding 1 mu L of the solution I into 1mL of RPMI1640 medium containing serum, fully and uniformly mixing, adding into the cell precipitate obtained in the step two, fully sucking the cell precipitate by using a pipette, re-suspending and uniformly mixing, and incubating for 15min in a cell culture box at 37 ℃.
Step four, cell fixation: directly adding 250 mu L of FI solution into the sample suspension, wherein the FI solution is specifically 5 multiplied by fixed and permeable solution (201065, Fluidigm), uniformly mixing, timing, incubating and fixing at room temperature, transferring the cell suspension in the fixation into a 15mL centrifugal tube, and marking;
step five, sealing: adding 50 μ L of liquid B to each sample to resuspend the cell pellet, and transferring to a clean 1.5mL EP tube, labeling, and incubating on ice; the solution B is prepared from 0.5-2mL human immunoglobulin solution (2mg/mL), 0.5-2mL mouse immunoglobulin solution (2mg/mL), 0.5-2mL rat immunoglobulin solution (2mg/mL), 0.5-2mL hamster immunoglobulin solution (2mg/mL), 0.5-2mg bovine serum albumin, and 100mL phosphate buffer (GNM 20012);
step six, staining the cell surface with antibodies: preparing a cell surface marker antibody mixed solution during the incubation period of a cell sample on ice, adding an antibody into a bovine serum albumin solution (0.5-2% by mass and volume, dissolved in a phosphate buffer solution) according to a proper dilution ratio, and adding 50 mu L of the antibody into each sample to prepare 50 mu L of the antibody mixed solution; adding 50 mu L of cell surface marker antibody mixed solution into each EP tube, mixing uniformly and incubating on ice;
step seven, transparent: adding 100 mu L of P solution into each cell sample to resuspend the cells, preparing two horizontal sections by 1mL of fixed/transparent concentrated solution (00-5123-43, Invitrogen) and 3mL of fixed/transparent diluted solution (00-5223-56, Invitrogen), and incubating at room temperature;
step eight, staining cell cycle antibodies: adding the solution C to 100 mu L of solution W, adding the solution C to the cell sediment, uniformly mixing, and incubating on ice; w solution is prepared from 1mL of 10X permeation solution (00-8333-56, Invitrogen) and 9mL of water;
step nine, cell secondary fixation: adding 1mL of F II solution into each EP tube, blowing and sucking by a pipette, uniformly mixing and resuspending cells, and incubating at room temperature; f II solution is composed of 0.9-1.3mL of 16 wt% formaldehyde solution and 9mL of phosphate buffer solution (GNM 20012);
step ten, DNA staining and fixing: adding 1mL of solution D into each EP tube, blowing and sucking by a pipette, uniformly mixing, suspending cells, and standing overnight at 0-12 ℃; the D liquid is prepared from 0.0005mLCell-IDTMIntercalator-Ir (201192B, Fluidigm), 0.09-0.13mL 16% (mass/volume ratio, mg/mL) formaldehyde solution, 0.9mL phosphate bufferPreparing a solution (GNM 20012);
step eleven, labeling cell barcodes: adding 100 mu L of BC solution containing phosphate buffer solution and any two combinations of Pd104, Pd105, Pd106, Pd108 and Pd110 into the sample cells, resuspending the cells, and incubating the cells on ice for 30 minutes;
step twelve, rinsing, and detecting on a machine: after rinsing the cells with double distilled water for 2 times, transferring the cells to a flow tube, counting the cells, and detecting the cells on a machine.
The invention has the following general beneficial effects:
the invention can realize specific, effective, high-throughput and single cell horizontal detection of the inner cell cycle state of the cell, greatly improves the depth and precision of sample detection, and greatly avoids the false negative analysis result possibly existing in the traditional detection means.
Specifically, the invention has the following beneficial effects:
(1) effectiveness: aiming at the antibodies in different cell cycles, the staining agent can effectively mark and distinguish the cell cycle state of the detected sample cell, and the cell cycle state is detected by ICP mass spectrometry to extract useful biological information contained in the cell cycle state;
(2) high throughput and single cell level: the invention realizes that the single cell level simultaneously detects and analyzes less than ten different types of sample cells and the number of cells with at least over 105 orders of magnitude, so that different sample cells can be maintained in the same sample processing condition and the same detection environment for monitoring and analysis, thereby avoiding the batch effect of the traditional flow cytometer for analyzing the experimental operation and the on-machine detection in the sample cell cycle, and improving the depth and the precision of the sample detection;
(3) avoiding false negative analysis results: the invention combines specific cell surface marker antibody staining, can detect the cell cycle state of the sample cell, and simultaneously carry out grouping identification on different types of cells in the sample cell, thereby identifying the possible cell subgroups of unknown types in the sample cell and whether the cell cycle state has difference, and avoiding the situation that the traditional flow cytometry detection means has difference on the cell cycle state of different sample cells and has no obvious difference after detection and analysis.
[ description of the drawings ]
FIG. 1 is a schematic flow diagram of a cell cycle detection method based on mass spectrometry flow detection technology;
FIG. 2 is a population of Jurkat cells in various cell cycle states;
FIG. 3 is a cell population of Jurkat cells in various cell cycle states;
FIG. 4 is a statistical result of cell cycle distribution ratio of Jurkat cells.
[ detailed description ] embodiments
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
The following examples are not intended to limit the scope of the present invention, nor are the steps described to limit the order in which they are performed, i.e., to aqueous solutions, unless otherwise specified, concentrations differing by unit from one type of concentration, e.g., "mg/mL" to indicate the ratio of solute mass to solution volume, and so on. Modifications of the invention which are obvious to those skilled in the art in view of the prior art are also within the scope of the invention as claimed.
A cell cycle detection kit based on a mass flow detection technology mainly comprises LD liquid, I liquid, FI liquid, FII liquid, B liquid, W liquid, P liquid, D liquid and BC1-BC10 liquid;
wherein the LD liquid comprises 194Pt and RPMI1640 basic culture medium; the solution I comprises iodoglycoside and dimethyl sulfoxide (DMSO); the F I liquid comprises a fixing liquid and a liquid penetrating liquid; f II liquid comprises formaldehyde and phosphate buffer solution; the solution B is a blocking buffer solution containing human immunoglobulin, mouse immunoglobulin, rat immunoglobulin, hamster immunoglobulin, bovine serum albumin and phosphate buffer solution and used for the flow cytometry; w liquid is a transparent buffer liquid; the liquid P is fixed/permeable liquid; the solution C is a cell cycle antibody staining solution; the solution D comprises 191/193Ir, formaldehyde and phosphate buffer solution; the BC1-BC10 liquid is a liquid comprising phosphate buffer and any two of Pd104, Pd105, Pd106, Pd108 and Pd 110.
The following examples also relate to the use of CD45(Biolegend), CD3e (Bioxcell), as cell surface antibody markers for sample cells, in the examples used to identify specific cell subpopulations of sample cell Jurkat cells.
The cell cycle detection method based on the mass flow detection technology comprises the following steps:
step one, preparing a sample single cell suspension:
1) collecting the Jurkat T cell single cell suspension obtained after digestion, and counting the cells;
2) get 107Unicellular, centrifugate 400g/5min at room temperature, abandon the supernatant;
3) at 107Adding a serum-free RPMI1640 culture medium preheated at 37 ℃ into the/mL cell density, centrifuging at room temperature for 400g/5min, and discarding the supernatant;
step two, determining cell death and cell viability;
4) adding 1mLLD solution (specifically 1 volume part of RPMI1640 basic medium, 0.01 volume part of 1mM194Pt (201194, Fluidigm)), gently blowing the cell sediment at the bottom of the tube to resuspend the cells; incubating in a cell culture box at 37 ℃ for 5 min;
5) adding 5mL RPMI1640 medium containing serum preheated at 37 deg.C, centrifuging at room temperature for 400g/5min, and discarding the supernatant;
step three, IdU incubation:
6) 1mL of RPMI1640 medium containing serum was added in the following ratio of 1: diluting solution I (specifically 0.354-35.4 parts by mass of iodoglycoside and 1 part by volume of dimethyl sulfoxide (Sigma)) at a ratio of 1000, and then resuspending the cells; 15min in a 37 ℃ cell culture box;
step four, cell fixation:
7) adding 250 μ L FI solution (specifically 5 × fixed and liquid permeable solution (201065, Fluidigm)) directly into the sample suspension, mixing, timing, and incubating at room temperature for 10 min; transferring the cell suspension in the fixation into a 15mL centrifugal tube, and marking;
8) adding 7.5mL of bovine serum albumin solution (0.5% by mass volume in phosphate buffer) into the cell suspension in each 15mL centrifuge tube to clean the cells, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant;
step five, sealing:
9) adding 50 μ L of liquid B to each sample to resuspend the cell pellet, transferring to a clean 1.5mL EP tube, labeling, and incubating on ice for 20 min;
step six, cell surface marker antibody:
10) preparing a cell surface marker antibody mixed solution during the incubation period of the cell sample on ice, adding antibodies CD3(1:100 volume ratio) and CD45(1:100 volume ratio) to a bovine serum albumin solution (0.5% by mass volume ratio, dissolved in a phosphate buffer) according to a proper dilution ratio, adding 50 mu L of each sample, and preparing 50 mu L of each sample;
11) adding 50 mu L of cell surface marker antibody mixed solution into each EP tube, uniformly mixing, and incubating on ice for 30 min;
12) adding 1mL of bovine serum albumin solution (0.5% by mass/volume in phosphate buffer) into each EP tube to wash the cells, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant;
step seven, transparent:
13) adding 1mL of W solution (specifically 1 volume part of 10 Xpermeant solution (00-8333-56, Invitrogen) and 9 volume parts of water) into each EP tube, centrifuging at 4 deg.C for 800g/5min, and discarding the supernatant;
14) adding 100 μ L of P solution (specifically 1 volume part of fixed/permeation concentrated solution (00-5123-43, Invitrogen) and 3 volume parts of fixed/permeation diluted solution (00-5223-56, Invitrogen)) into each cell sample, and incubating at room temperature for 30 min;
15) adding 1mL of W solution into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant for the first time;
16) adding 1mL of W solution into each EP tube, centrifuging at 4 ℃ for 800g/5min, and discarding the supernatant for the second time;
step eight, staining cell cycle antibodies:
17) adding 100 μ LC solution (specifically, antibody incubation solution prepared from 1 volume part of CyclinB1-153Eu (1:100, Fluidigm), 1 volume part of phosphorus-Histone H3-165Ho (1:100, Biolegend), 1 volume part of phosphorus-Rb-150 Nd (1:100, Fluidigm), 0.5-2 mass parts of bovine serum albumin, and 97 volume parts of phosphate buffer solution (GNM20012)) into sample cells, mixing, and incubating on ice for 30 min;
18) adding 1mLP (specifically 1 volume part of fixed/transparent concentrated solution (00-5123-43, Invitrogen) and 3 volume parts of fixed/transparent diluent (00-5223-56, Invitrogen)) into each EP tube to stop washing cells, centrifuging at 4 ℃ for 800g/5min, and discarding the supernatant;
19) adding 1mL of phosphate buffer solution (GNM20012) into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing supernatant;
step nine, cell secondary fixation:
20) adding 1mL of FII solution (specifically, 0.9-1.3 volume parts of 16 wt% formaldehyde solution and 9 volume parts of phosphate buffer solution (GNM20012)) into each EP tube, blowing and sucking by a pipette, uniformly mixing and resuspending cells, and incubating at room temperature for 10 min;
21) centrifuge at 4 ℃ for 800g/5min and discard the supernatant.
Step ten, DNA staining and fixing:
22) to each EP tube, 1mL of D solution (specifically, 0.0005 part by volume of Cell-ID) was addedTMIntercalator-Ir (201192B, Fluidigm), 0.09-0.13 parts by volume of 16 wt% formaldehyde solution, 0.9 parts by volume of phosphate buffer (GNM20012)), and a pipette gun to suck, mix, resuspend the cells, and place the cells in a refrigerator at 4 ℃ for overnight.
Step eleven, labeling cell barcodes:
23) taking out the cell sample from a refrigerator at 4 ℃, centrifuging at 4 ℃ for 800g/5min, and removing the supernatant;
24) adding 1mL of phosphate buffer solution (GNM20012) into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing supernatant;
25) adding 100 μ L of BC1 solution (containing 1 volume part of Pd104(1.5-2 μ M, Fluidigm), 1 volume part of Pd105(1.5-2 μ M, Fluidigm), 100 volume parts of phosphate buffer (GNM20012)) to the sample cells, resuspending the cells, and incubating on ice for 30 min;
26) adding 1mL of bovine serum albumin solution (0.5-2 parts by mass of bovine serum albumin, 100 parts by volume of phosphate buffer GNM20012, wherein the parts by mass are mg and the parts by volume are mL) into each EP tube, centrifuging at 4 ℃ for 800g/5min, and removing supernatant;
27) adding 1mL of ultrapure water into each EP tube to resuspend cells, centrifuging at 4 ℃ for 800g/5min, and discarding the supernatant;
28) preparing a flow pipe with a filter membrane filter head, and marking the pipe wall;
step twelve, rinsing, and detecting on a machine:
29) adding 1mL of ultrapure water into each EP tube to resuspend cells, sucking cell suspension by using a pipette gun, transferring the cell suspension to a corresponding flow tube through a filter head filter membrane of the flow tube, adding 1mL of ultrapure water again to clean the wall of the EP tube for a plurality of times, sucking cleaning liquid by using the pipette gun, filtering the same flow tube filter membrane, and putting the same flow tube into the same flow tube;
30) centrifuging at 4 deg.C for 800g/5min, and removing supernatant;
31) adding 1mL of ultrapure water into each flow tube to resuspend cells, and respectively taking 10 mu L of the cell to count the cells;
32) according to the cell counting result, the cells of the control group and the treated group with the same cell number are taken, after mixing, the cell sample is washed for a plurality of times by water, a proper amount of balance magnetic beads are added, the detection is carried out on a Helios mass spectrometer (Fluidigm) machine, and the two groups of data are processed and analyzed.
The results are shown in fig. 2, 3 and 4. The Jurkat cell line was extracellularly stained with CD45 and CD3 antibodies, and a T cell population was isolated with a cell proportion of 46.4% of the total cell number (FIG. 2). Meanwhile, after cell cycle detection using a cell cycle detection kit based on mass spectrometry flow detection technology, we succeeded in isolating a cell population in which the T cell population of Jurkat cells was in each cell cycle state (fig. 3). Based on the statistical number of the cell cycle cell populations circled, the cell cycle distribution ratio of Jurkat cells was obtained (FIG. 4). For the test sample Jurkat cell line of this example, the cell number ratios of the T cell subsets in the G0, G1, S, G2, M cycles were 3.59%, 48.45%, 41.33%, 5.62%, and 1.00%, respectively.

Claims (13)

1. The cell cycle detection kit based on the mass flow detection technology is characterized by comprising LD liquid, I liquid, FI liquid, FII liquid, B liquid, W liquid, P liquid, C liquid, D liquid and BC1-BC10 liquid;
the LD liquid comprises 194Pt and RPMI1640 basic culture medium;
the liquid I comprises iodoglycoside and dimethyl sulfoxide;
the F I liquid comprises a fixed liquid and a permeable liquid;
the F II solution is formaldehyde and phosphate buffer solution;
the solution B is a blocking buffer solution which contains human immunoglobulin, mouse immunoglobulin, rat immunoglobulin, hamster immunoglobulin, bovine serum albumin and phosphate buffer solution and is used for flow cytometry;
the W liquid is a transparent buffer liquid;
the liquid P is a fixed/permeable liquid;
the solution C is a cell cycle antibody staining solution;
the solution D comprises 191/193Ir, formaldehyde and phosphate buffer solution;
the BC1-BC10 liquid is a liquid comprising phosphate buffer and any two of Pd104, Pd105, Pd106, Pd108 and Pd 110.
2. The cell cycle detection kit based on mass spectrometry flow detection technology of claim 1, wherein the LD solution is 1 volume part of RPMI1640 basic medium, 0.01 volume part of 1mM194 Pt.
3. The cell cycle detection kit based on the mass spectrometry flow detection technology as claimed in claim 1, wherein the mass-to-volume ratio of the iodoglycoside and the dimethyl sulfoxide in the solution I is (0.354-35.4): 1, wherein the mass is in mg units and the volume is in mL units.
4. The cell cycle detection kit based on mass spectrometry flow detection technology as claimed in claim 1, wherein the FI fluid is 5 x fixed and permeable fluid.
5. The cell cycle detection kit based on mass spectrometry flow detection technology of claim 1, wherein the F II solution is 0.9-1.3 parts by volume of 16 wt% formaldehyde solution and 9 parts by volume of phosphate buffer solution.
6. The cell cycle detection kit based on mass flow detection technology of claim 1, wherein the solution B is 0.5-2 parts by volume of a human immunoglobulin solution with a concentration of 2mg/mL, 0.5-2 parts by volume of a mouse immunoglobulin solution with a concentration of 2mg/mL, 0.5-2 parts by volume of a rat immunoglobulin solution with a concentration of 2mg/mL, 0.5-2 parts by volume of a hamster immunoglobulin solution with a concentration of 2mg/mL, 0.5-2 parts by mass-to-volume ratio of bovine serum albumin, 100 parts by volume of a phosphate buffer; wherein the mass-to-volume ratio is the ratio of the mass of bovine serum albumin in mg to the volume of liquid B in mL.
7. The cell cycle detection kit based on mass spectrometry flow detection technology of claim 1, wherein the W liquid is 1 volume part of 10 x permeable liquid and 9 volume parts of water.
8. The cell cycle detection kit based on mass spectrometry flow detection technology as claimed in claim 1, wherein the liquid P is 1 volume part of fixed/permeation concentrated liquid and 3 volume parts of fixed/permeation diluted liquid.
9. The cell cycle detection kit based on the mass spectrometry flow detection technology of claim 1, wherein the solution C is an antibody staining solution prepared from 1 volume part of Cyclin B1-153Eu, 1 volume part of phosphorus-Histone H3-165Ho, 1 volume part of phosphorus-Rb-150 Nd, 0.5-2 parts by mass of bovine serum albumin, and 97 volume parts of phosphate buffer; wherein the mass-to-volume ratio is the ratio of the mass of bovine serum albumin in mg to the volume of liquid C in mL.
10. The mass spectrometry flow detection technology-based cell cycle detection assay of claim 1A kit, wherein the D liquid is 0.0005 volume part of Cell-IDTMIntercalator-Ir, 0.09-0.13 volume parts of 16 wt% formaldehyde solution and 0.9 volume parts of phosphate buffer solution.
11. The cell cycle detection kit based on mass spectrometry flow detection technology as claimed in claim 1, wherein the BC1 fluid contains 1 part by volume of 1.5-2 μ M Pd104, 1 part by volume of 1.5-2 μ M Pd105, and 100 parts by volume of phosphate buffer;
the BC2 solution contains 1 volume part of 1.5-2 mu M Pd106, 1 volume part of 1.5-2 mu M Pd108 and 100 volume parts of phosphate buffer solution;
the BC3 solution contains 1 volume part of 1.5-2 mu M Pd105, 1 volume part of 1.5-2 mu M Pd110 and 100 volume parts of phosphate buffer solution;
the BC4 solution contains 1 volume part of 1.5-2 mu M Pd104, 1 volume part of 1.5-2 mu M Pd106 and 100 volume parts of phosphate buffer solution;
the BC5 solution contains 1 volume part of 1.5-2 mu M Pd105, 1 volume part of 1.5-2 mu M Pd108 and 100 volume parts of phosphate buffer solution;
BC6 liquid contains 1 volume part of 1.5-2 μ M Pd106, 1 volume part of 1.5-2 μ M Pd110, 100 volume parts of phosphate buffer;
the BC7 solution contains 1 volume part of 1.5-2 mu M Pd104, 1 volume part of 1.5-2 mu M Pd108 and 100 volume parts of phosphate buffer solution;
the BC8 solution contains 1 volume part of 1.5-2 mu M Pd105, 1 volume part of 1.5-2 mu M Pd106 and 100 volume parts of phosphate buffer solution;
the BC9 solution contains 1 volume part of 1.5-2 mu M Pd104, 1 volume part of 1.5-2 mu M Pd110 and 100 volume parts of phosphate buffer solution;
the BC10 solution contains 1 volume part of 1.5-2 μ M Pd108, 1 volume part of 1.5-2 μ M Pd110, and 100 volume parts of phosphate buffer solution.
12. The cell cycle detection kit based on mass spectrometry flow detection technology of claim 1, wherein the solution LD, solution I, solution FI, solution FII, solution B, solution W, solution P, solution C, solution D, and solution BC1-BC10 are stored at 2-8 ℃ after being prepared on ice.
13. A method of cell cycle detection based on mass spectrometric detection, characterized in that the use of a kit according to any one of claims 1 to 12 comprises the following steps:
step one, preparing a sample single cell suspension: collecting single cell suspension, each sample containing (1-5). times.106The cell suspension of individual cell amount was centrifuged at room temperature, and the supernatant was carefully discarded to obtain a cell pellet.
Step two, determining cell death and cell viability: adding 1mL of LD solution, gently sucking the cell precipitate by a pipette to fully resuspend the cell precipitate, and placing the cell precipitate in a cell culture box at 37 ℃ for incubation; after addition of 5mL of serum-free RPMI1640 medium, the mixture was centrifuged at room temperature, and the supernatant was carefully discarded to obtain a cell pellet.
Step three, IdU incubation: adding 1 mu L of the solution I into 1mL of RPMI1640 medium containing serum, fully and uniformly mixing, adding into the cell precipitate obtained in the step two, fully sucking the cell precipitate by using a pipette, re-suspending and uniformly mixing, and placing into a cell culture box at 37 ℃ for incubation;
step four, cell fixation: directly adding 250 mu L of FI solution into the sample suspension, wherein the FI solution is 5 multiplied by fixed solution and permeable solution, uniformly mixing, timing, incubating at room temperature, transferring the cell suspension in the fixation into a centrifugal tube, and marking;
step five, sealing: add 50. mu.L of B liquid to each sample to resuspend the cell pellet and transfer to a clean EP tube, labeled, incubated on ice;
step six, staining the cell surface with antibodies: preparing a cell surface marker antibody mixed solution during the incubation period of the cell sample on ice, adding the antibody into a bovine serum albumin solution according to a proper dilution ratio, and preparing 50 mu L of each sample; adding 50 mu L of cell surface marker antibody mixed solution into each EP tube, mixing uniformly and incubating on ice;
step seven, transparent: adding 100 mu L of P liquid into each cell sample to resuspend the cells, and incubating at room temperature;
step eight, staining cell cycle antibodies: adding the solution C to 100 mu L of solution W, adding the solution C to the cell sediment, uniformly mixing, and incubating on ice;
step nine, cell secondary fixation: adding 1mL of F II solution into each EP tube, blowing and sucking by a pipette, uniformly mixing and resuspending cells, and incubating at room temperature; f II solution consists of 0.9-1.3mL of 16 wt% formaldehyde solution and 9mL of phosphate buffer solution;
step ten, DNA staining and fixing: adding 1mL of solution D into each EP tube, blowing and sucking by a pipette, uniformly mixing, suspending cells, and standing at 0-12 ℃ overnight;
step eleven, labeling cell barcodes: adding 100 μ L of any one of BC1-BC10 solution into the sample cells, resuspending the cells, and incubating on ice;
step twelve, rinsing, and detecting on a machine: after rinsing the cells with water, the cells were transferred to a flow tube and counted and tested on a machine.
CN201910846725.5A 2019-09-09 2019-09-09 Cell cycle detection kit based on mass flow detection technology and detection method Active CN110608991B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910846725.5A CN110608991B (en) 2019-09-09 2019-09-09 Cell cycle detection kit based on mass flow detection technology and detection method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910846725.5A CN110608991B (en) 2019-09-09 2019-09-09 Cell cycle detection kit based on mass flow detection technology and detection method

Publications (2)

Publication Number Publication Date
CN110608991A true CN110608991A (en) 2019-12-24
CN110608991B CN110608991B (en) 2022-04-29

Family

ID=68892431

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910846725.5A Active CN110608991B (en) 2019-09-09 2019-09-09 Cell cycle detection kit based on mass flow detection technology and detection method

Country Status (1)

Country Link
CN (1) CN110608991B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111189761A (en) * 2020-01-10 2020-05-22 浙江普罗亭健康科技有限公司 Cell signal channel detection kit based on mass spectrometry flow technology and detection method
CN113125733A (en) * 2021-04-16 2021-07-16 浙江普罗亭健康科技有限公司 42 antibody kit for monitoring human immune state and application thereof
CN114441417A (en) * 2022-01-18 2022-05-06 重庆生命知源科技有限公司 Flow cytometer based detection method for trace cell phosphorylation protein expression
CN117757730A (en) * 2023-12-26 2024-03-26 浙江普罗亭健康科技有限公司 Kit special for digestion of human heart tissue, digestion method and detection method

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160201031A1 (en) * 2013-08-21 2016-07-14 The Regents Of The University Of California Composition and methods for culturing cells
US20160379811A1 (en) * 2015-06-24 2016-12-29 City University Of Hong Kong Method of determining cell cycle stage distribution of cells
US20170059574A1 (en) * 2015-08-25 2017-03-02 The Board Of Trustees Of The Leland Stanford Junior University Platinum-labeled probes for mass cytometry
CN106535934A (en) * 2014-04-10 2017-03-22 西雅图儿童医院（Dba西雅图儿童研究所） Defined composition gene modified t-cell products
CN107299128A (en) * 2017-08-03 2017-10-27 北京多赢时代转化医学研究院 A kind of kit and method for detecting intracellular ALDH activity
CN107656044A (en) * 2017-09-25 2018-02-02 亚能生物技术（深圳）有限公司 The detection kit and detection method of a kind of circulating tumor cell
CN107796748A (en) * 2017-09-28 2018-03-13 上海交通大学 A kind of detection method for unicellular mass spectrum flow cytometry
CN108226016A (en) * 2018-01-12 2018-06-29 浙江普罗亭健康科技有限公司 The mass spectrum flow cytometer detection kit of the accurate parting of tumor vaccine cells subgroup
CN108508196A (en) * 2018-04-13 2018-09-07 沈阳汇敏源生物科技有限责任公司 A kind of kit and detection method of detection people's regulatory T cells hypotype
CN109323908A (en) * 2018-10-10 2019-02-12 浙江大学 A kind of detection method for mass spectrum streaming technology

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160201031A1 (en) * 2013-08-21 2016-07-14 The Regents Of The University Of California Composition and methods for culturing cells
CN106535934A (en) * 2014-04-10 2017-03-22 西雅图儿童医院（Dba西雅图儿童研究所） Defined composition gene modified t-cell products
US20160379811A1 (en) * 2015-06-24 2016-12-29 City University Of Hong Kong Method of determining cell cycle stage distribution of cells
US20170059574A1 (en) * 2015-08-25 2017-03-02 The Board Of Trustees Of The Leland Stanford Junior University Platinum-labeled probes for mass cytometry
CN107299128A (en) * 2017-08-03 2017-10-27 北京多赢时代转化医学研究院 A kind of kit and method for detecting intracellular ALDH activity
CN107656044A (en) * 2017-09-25 2018-02-02 亚能生物技术（深圳）有限公司 The detection kit and detection method of a kind of circulating tumor cell
CN107796748A (en) * 2017-09-28 2018-03-13 上海交通大学 A kind of detection method for unicellular mass spectrum flow cytometry
CN108226016A (en) * 2018-01-12 2018-06-29 浙江普罗亭健康科技有限公司 The mass spectrum flow cytometer detection kit of the accurate parting of tumor vaccine cells subgroup
CN108508196A (en) * 2018-04-13 2018-09-07 沈阳汇敏源生物科技有限责任公司 A kind of kit and detection method of detection people's regulatory T cells hypotype
CN109323908A (en) * 2018-10-10 2019-02-12 浙江大学 A kind of detection method for mass spectrum streaming technology

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GREGORY K. BEHBEHANI 等: "Single-Cell Mass Cytometry Adapted to Measurements of the Cell Cycle", 《CYTOMETRY PART A》 *
QING CHANG 等: "Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry", 《INTERNATIONAL JOURNAL OF CANCER》 *
周琰 等: "临床 （前） 研究的质谱流式细胞术———流式细胞技术的 "后荧光时代"", 《中国新药与临床杂志》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111189761A (en) * 2020-01-10 2020-05-22 浙江普罗亭健康科技有限公司 Cell signal channel detection kit based on mass spectrometry flow technology and detection method
CN113125733A (en) * 2021-04-16 2021-07-16 浙江普罗亭健康科技有限公司 42 antibody kit for monitoring human immune state and application thereof
CN114441417A (en) * 2022-01-18 2022-05-06 重庆生命知源科技有限公司 Flow cytometer based detection method for trace cell phosphorylation protein expression
CN114441417B (en) * 2022-01-18 2023-02-07 重庆生命知源科技有限公司 Flow cytometer based detection method for trace cell phosphorylation protein expression
CN117757730A (en) * 2023-12-26 2024-03-26 浙江普罗亭健康科技有限公司 Kit special for digestion of human heart tissue, digestion method and detection method
CN117757730B (en) * 2023-12-26 2024-06-07 浙江普罗亭健康科技有限公司 Kit special for digestion of human heart tissue, digestion method and detection method

Also Published As

Publication number Publication date
CN110608991B (en) 2022-04-29

Similar Documents

Publication Publication Date Title
CN110608991B (en) Cell cycle detection kit based on mass flow detection technology and detection method
CN110567861B (en) Kit for screening antigenic peptide with immunogenicity based on mass flow detection technology and detection method
CN101587043B (en) Integrated method for enriching and detecting rare cell in biological fluid sample
CN105954246B (en) Method and kit for detecting free rare tumor cells in human biological fluid sample
JP7124062B2 (en) Method for detecting the presence of an analyte in a urine sample
CN111189761A (en) Cell signal channel detection kit based on mass spectrometry flow technology and detection method
US5242806A (en) Method for conducting the cytotoxicity assays on tumor cells
EP3712599B1 (en) Method for detecting and typing rare tumor cells in body fluid sample and kit therefor
CN111527406A (en) Preparation method of lymphocyte sample for flow cytometry analysis
CN110361442B (en) Exosome for mass cytometry detection and preparation method and application thereof
CN115166252A (en) Lymphocyte subset grouping and quantitative detection kit, detection method and application thereof
CN108152189B (en) Quantitative detection method for sperm surface clouding protein 1
CN102313813B (en) Integration method for enriching and detecting rare cells from biological fluid samples
CN112946264A (en) Sample pretreatment method and immune cell detection method for flow cytometry
CN110702909A (en) Negative enrichment detection method for circulating tumor cells
US20240011989A1 (en) Method for identification of viruses and diagnostic kit using the same
CN111528219B (en) Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof
CN116296702B (en) Dead-living dye capable of customizing detection channel based on mass spectrometry detection technology and preparation method and application thereof
US8728751B2 (en) System and method for diagnosing lymphoma in cats
CN114487394A (en) Cell cycle detection kit and application thereof
CN117214438A (en) Cell five-period detection kit and application
CN116678810A (en) Cell proliferation immunofluorescence detection kit and application thereof
CN117025741A (en) Method for controlling effective quality of amniotic membrane-derived mesenchymal stem cells
WO1992009704A1 (en) Method for in situ detection and identification of nucleic acid sequences
US20040166540A1 (en) Methods of detecting CD34 positive and negative hematopoietic stem cells in human samples

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
PE01 Entry into force of the registration of the contract for pledge of patent right
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: Cell cycle detection kit and detection method based on mass spectrometry flow cytometry technology

Effective date of registration: 20230830

Granted publication date: 20220429

Pledgee: Zhejiang Hangzhou Yuhang Rural Commercial Bank Co.,Ltd. Science and Technology City Branch

Pledgor: ZHEJIANG PLTTECH HEALTH TECHNOLOGY CO.,LTD.

Registration number: Y2023980054407