CN111157433A - Kit for marking single cell of red blood cell and detection method thereof - Google Patents
Kit for marking single cell of red blood cell and detection method thereof Download PDFInfo
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Images
Classifications
-
- 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—Electro-optical investigation, e.g. flow cytometers
-
- 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
- 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
Abstract
The invention relates to a staining solution for marking cells in mass cytometry, a kit containing the staining solution, a detection method and an erythrocyte shaping solution. The staining solution contains osmate, and the kit further comprises a cell-specific metal-labeled antibody. By combining with the erythrocyte shaping solution, the kit is particularly suitable for the marking detection of single erythrocyte and can be used for the distribution determination of single cells of various elements in the erythrocyte.
Description
Technical Field
The invention relates to a staining solution for cell labeling in mass cytometry, a kit containing the staining solution and a detection method using the kit. The staining solution, the kit and the detection method are particularly suitable for measuring the distribution of various elements of single cells of anucleated cells, particularly red blood cells.
Background
Mass spectrometry by time-of-flight (CyTOF) is a single cell detection technology which develops rapidly in recent years, and is a flow cytometer based on the mass spectrometry detection principle, which adopts metal elements iridium (Ir) or rhodium (Rh) to mark cell nuclei for screening and identifying single cells; the method adopts non-radioactive rare earth elements as antibody labels, has accurate signals, a plurality of detection channels and high stability, and can simultaneously detect the information of at most 135 elements in a single cell compared with the traditional mass spectrometer because the overlapping interference of spectrum correction is not needed, thereby being an effective research means.
Erythrocytes are the main cell type of blood cells, have functions of transporting oxygen and carbon dioxide, immunoadhesion, immunophagy and the like, play a certain buffering role in acid-base balance, and are very important for maintaining normal physiological activities of a living body.
The element content in the red blood cells reflects the biological accumulation of the elements and the transfer and transformation rules of the elements in the human body, and is closely related to the health of the human body. The distribution of element content has individual heterogeneity among single red blood cells, part of red blood cells have high element content, part of red blood cells have low element content, and the element content is closely related to the function and health effect of the red blood cells. The analysis of the element content in single erythrocyte has very important significance for researching the distribution rule, the transfer transformation rule and relevant physiological and pathological conditions.
At present, the detection of the element content in the red blood cells is limited to total amount detection, and the detection methods mainly include ultraviolet visible spectrophotometry (UV), Atomic Absorption Spectroscopy (AAS), inductively coupled plasma emission spectrometer (ICP-OES), inductively coupled plasma mass spectrometry (ICP-MS), and the like. Therefore, there is a problem that only the average element content of red blood cells can be analyzed, and the difference in element content between individual red blood cells cannot be analyzed.
When single cell detection is carried out on erythrocytes, the main obstacle exists that the erythrocytes do not have cell nuclei, so that the erythrocytes cannot be marked by the metal elements iridium (Ir) or rhodium (Rh) used in the existing mass spectrometer; in addition, the red blood cells are characterized by easy rupture, and the red blood cells are very easy to rupture due to the change of the osmotic pressure inside and outside the cells, so that the complete cell morphology in the detection process is difficult to ensure.
In practice, it is often difficult to perform mass cytometry measurements at the first time after blood collection, depending on sample collection conditions, test equipment arrangement, parallel test scheduling, and the like. Therefore, it is desirable to have a reagent and method that can preserve a red blood cell sample for a long period of time while maintaining morphological integrity.
In view of the above, there is a need for new technical support for realizing element detection in single red blood cells, and long-term preservation agents which can be used in scientific research and commercial industries for keeping red blood cells intact.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a staining solution, a kit and a detection method thereof, which are suitable for detecting single cell elements of anucleated cells, particularly erythrocytes. The kit can realize the in-vitro long-term preservation of the red blood cells and is suitable for single cell element detection of a mass spectrum flow cytometer aiming at the red blood cells.
Osmate is a lipid reactive agent that can be used for preparation of transmission electron microscopy pathological diagnosis samples by staining cell membranes well using osmate fixative when observing tissue cells using transmission electron microscopy. In such applications, the concentration of osmic acid used is generally 2% (w/w), and sometimes 1% (w/w) is also used.
In flow cytometry, the labeling amount of the element osmium is strictly related to the size of a labeled cell, so that the signal of the element osmium can be used for distinguishing a whole single cell from cell fragments, and the detection of the single cell is realized. The inventors have found that the use of a catalyst having a concentration of 10 at the working concentration-5％～10-4% (w/v), preferably 3X 10-5％～7×10-5% (w/v), more preferably 5X 10-5％～7×10-5% (w/v) osmate solution as staining solution, good staining of cell samples, especially erythrocytes, was obtained in mass flow cytometry assay at single cell level.
Mass cytometry uses lanthanide metals, which are present in very low levels in cells, to label antibodies instead of traditional fluorescent proteins. Such an antibody labeled with a lanthanide is sometimes referred to herein simply as a metal-labeled antibody, wherein the metal-labeled antibody that binds to a specific cell (hereinafter referred to as a cell-specific metal-labeled antibody) is capable of specifically binding to the specific cell, thereby enabling screening and identification of the specific cell, for example, an erythrocyte-specific metal-labeled antibody can identify erythrocytes, distinguish erythrocytes from other cell types. When the lanthanide in the lanthanide-labeled antibody used in common is different from the lanthanide in the detection, 30 or even 40 kinds of antibodies can be used simultaneously for labeling. Herein, the capital letter M is sometimes used to represent the lanthanide.
The staining solution of the present invention can be used in combination with a metal-labeled antibody to favorably identify a target cell and label a single cell, thereby detecting the target cell at a single cell level. The staining solution for detecting a cytoelement of the present invention may be used in combination with other cell differentiation markers commonly used in the art, as long as the function of differentiating intact cells from cell debris can be utilized to successfully label the level of a single cell.
As metal-labeled antibodies, cell-specific metal-labeled antibodies, particularly erythrocyte-specific metal-labeled antibodies are used in the present invention, including but not limited to: at least one of an anti-mouse Ter 119-lanthanide-labeled antibody, an anti-human CD235 ab-lanthanide-labeled antibody, an anti-mouse CD 45-lanthanide-labeled antibody, or an anti-human CD 45-lanthanide-labeled antibody.
The lanthanoid used in the present invention may be, for example, one or more of La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), scandium (Sc), and yttrium (Y), and it is to be noted that, in the detection, lanthanoid in a lanthanoid-labeled antibody used in common needs to be different from each other.
The present invention also provides a erythrocyte fixative solution which can preserve erythrocytes in vitro, preferably at low temperature, more preferably at-80 ℃, for a long period of time, comprising a glutaraldehyde solution at a concentration of 2% to 5% (v/v), preferably 2% to 3% (v/v), particularly preferably 2.5% (v/v).
The in vitro long-term preservation refers to the following steps: after the preservation, the preserved cells may be used for the next measurement of biochemical indices of cells or the like by observing the morphology of intact cells with a scanning electron microscope or other techniques in the art, and the cells do not necessarily have to be living cells.
The "single cell assay" of the present invention includes the simultaneous or batch detection of one or more indicators of a cell population, resulting in multiple or single elemental content, biochemical, immunological and multiple information about individual (individual) cells in the cell population.
Specifically, the present invention relates to the following:
1. a staining solution for labeling cells (e.g., erythrocytes or leukocytes) in mass cytometry comprising osmate at a working concentration of the staining solution of 10-5％～10-4% (w/v), preferably 3X 10-5％～7×10-5% (w/v), more preferably 5X 10-5％～7×10-5％(w/v)。
2. A kit comprising the staining solution of item 1, further comprising: the metal-labeled antibody is preferably a cell-specific metal-labeled antibody.
3. The kit according to item 1 or 2, wherein: when the cells are red blood cells, the kit further comprises a red blood cell shaping solution, and the red blood cell shaping solution contains glutaraldehyde with the concentration of 2-5% (v/v), preferably 2-3% (v/v).
4. The kit according to any one of items 2 to 3, wherein the cell-specific metal-labeled antibody is a lanthanide-labeled antibody (preferably one or more of an anti-mouse Ter 119-lanthanide-labeled antibody, an anti-human CD235 ab-lanthanide-labeled antibody, an anti-mouse CD 45-lanthanide-labeled antibody, or an anti-human CD 45-lanthanide-labeled antibody), and optionally, lanthanides in the lanthanide-labeled antibodies used in common are different from each other at the time of detection.
5. The kit according to item 4, wherein the lanthanide in the lanthanide-labeled antibody is selected from La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), scandium (Sc), yttrium (Y), preferably Sm (samarium), Pr (praseodymium).
6. The kit according to any one of claims 2 to 5, further comprising a buffer for staining, wherein the buffer for staining comprises 0.5 to 1% (w/v) of a blocking protein (such as bovine serum albumin) and is a buffer free of calcium and magnesium ions (preferably a phosphate buffer having a pH range of 7.2 to 7.4, and more preferably PBS).
7. The kit according to item 2 to 6, further comprising a cell washing solution, wherein the cell washing solution (preferably phosphate buffer solution with pH ranging from 7.2 to 7.4, more preferably PBS) does not contain calcium and magnesium ions, and preferably the concentration of the phosphate buffer solution without the magnesium ions is 0.0067M.
8. Use of the kit according to any one of items 2 to 7 for detecting a single cell, wherein the cell to be detected preferably comprises red blood cells.
9. Application of glutaraldehyde solution with concentration of 2-5% (v/v) in long-term preservation of erythrocytes.
10. A method of single-cell elemental detection comprising the steps of:
treating the cells with a glutaraldehyde solution at a concentration of 2% to 5% (v/v), preferably 2% to 3% (v/v);
using osmic acid in a concentration of 10-510-4％(w/v), preferably 3X 10-5～7×10-5% (w/v), more preferably 5X 10-5～7×10-5% w/v of the staining solution for labeling the cells (preferably, the concentration of the cells is 2X 106～4×106One/ml);
the cells are labeled with a metal-labeled antibody, preferably a cell-specific metal-labeled antibody.
11. The method of item 10, further comprising one or more of the following steps:
dissolving the metal-labeled antibody in a staining buffer as defined in item 6;
washing the cells with a staining buffer as defined in item 6;
the cells were washed using the cell wash defined in item 7.
The staining solution, the kit and the detection method thereof provided by the invention are particularly suitable for detecting the single cell elements of anucleated cells, particularly erythrocytes, and can realize the morphological fixation, specific screening and erythrocyte membrane marking of the erythrocytes by utilizing an osmate solution as the staining solution, a glutaraldehyde solution as an erythrocyte shaping solution and an erythrocyte specific metal marking antibody.
It should be noted that although osmium tetroxide solution is commonly used as a fixing solution in the field of cell detection, the concentration of osmic acid in the solution used in the present invention is much lower than that of osmium tetroxide used for cell fixation, which is not an order of magnitude, and the properties of the utilized osmium are not the same.
Drawings
FIG. 1 is a graph showing the integrity of red blood cells treated with the erythrocyte fixative solution of the present invention. Wherein the content of the first and second substances,
FIG. 1a shows the results after treatment with glutaraldehyde and Paraformaldehyde (PFA) solutions, respectively, the glutaraldehyde solution on the left and the paraformaldehyde solution on the right;
FIG. 1b is a scanning electron microscopic morphology of glutaraldehyde solution-fixed red blood cells;
FIG. 1c is a bright field configuration under a laser scanning confocal microscope of glutaraldehyde solution-fixed red blood cells;
FIG. 1d is a graph showing fluorescence (Ter119-PE) patterns observed by a laser scanning confocal microscope on red blood cells fixed to a glutaraldehyde solution.
FIG. 2 shows the effect of osmate labeling at different concentrations, wherein FIG. 2a shows the ratio of single cells detected by mass cytometry after osmate labeling at different concentrations; FIGS. 2 b-2 h show the concentration of 5X 10-6％、10-5％、3×10-5％、5×10-5％、7×10-5％、10-4％、5×10-5% (w/v) osmate-labeled single cell two-dimensional scattergrams.
FIG. 3 is a sample of red blood cells after treatment with a red blood cell fixative wherein (a) is the proportion of single cells in the sample that are preserved for 15 days and 150 days after treatment with the red blood cell fixative; (b) and (c) two-dimensional scatter plots of single cells stored for 15 days and 150 days, respectively, (d) - (h) elements78Se、88Sr、127I、208Pb、209Distribution profile of Bi in single cells.
FIG. 4 is a comparison of the results of nucleated cell detection using the kit and method of the present invention and the iridium labeling method. Wherein, (a) is a white blood cell ratio chart marked by utilizing an iridium element and the kit of the invention; (b) is a proportion chart of cells obtained by labeling lead-containing white blood cells (exposure group) after being exposed to lead by using an iridium element and the kit of the invention.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with specific embodiments.
As an aspect of the present invention, there is provided a use of a glutaraldehyde solution for long-term preservation of red blood cells, and a method for preserving red blood cells using the same, for example, includes the steps of:
adding fresh human or animal anticoagulated whole blood into a phosphate buffer solution (without calcium and magnesium ions) with the volume being 3 times that of the human or animal anticoagulated whole blood, and uniformly mixing to obtain a single cell suspension;
dropwise adding a glutaraldehyde solution with the concentration of 2-5% (v/v) which is 4 times of the volume of the cell suspension into the cell suspension, and standing the cell suspension at normal temperature for 10-30 min;
washing the cells by centrifugation with phosphate buffer (without calcium and magnesium ions);
cell cryopreservation: suspending the cells in a buffer solution containing 1-3% (w/v) of blocking protein (such as bovine serum albumin) and no calcium and magnesium ions, or a solution commonly used in the art for cell cryopreservation, and freezing at-80 deg.C in a refrigerator.
As another aspect of the present invention, there is provided a single cell detection kit and a method using the same, the method comprising the steps of:
adding fresh human or animal anticoagulated whole blood into a phosphate buffer solution (without calcium and magnesium ions) with the volume being 3 times that of the human or animal anticoagulated whole blood, and uniformly mixing to obtain a single cell suspension;
dropwise adding the erythrocyte shaping solution into the cell suspension, and standing for 10-30 min at normal temperature;
washing the cells by centrifugation with phosphate buffer (without calcium and magnesium ions);
it should be noted that, although 2 × 10 is used herein6～4×106Individual cell, but at a cell concentration of 1X 106～8×106The effect of the present invention can be achieved within the range of one/ml;
osmic acid solution labeling: the cells were all suspended in 100. mu.l of staining buffer, and 1ml of 10 cells were added-5～10-4Percent (w/v) osmic acid solution while continuously vortexing the cell suspension, standing at normal temperature for 10-30 min, and then washing the cells by centrifugation using staining buffer;
after washing the cells 3 times by centrifugation with ultrapure water, detection of elements in individual erythrocytes was completed by a mass flow cytometer.
The erythrocyte fixative is a reagent that can fix erythrocytes without rupturing erythrocytes, and is preferably a glutaraldehyde solution having a concentration of 2% to 5% (v/v), more preferably 2% to 3% (v/v).
Further, the staining solution, the kit and the erythrocyte typing solution of the present invention are preferably stored at a low temperature, and the storage condition is, for example, 2 to 8 ℃.
Dispersing: it is preferable to disperse the cells by vortexing, beating, or the like when a erythrocyte fixative solution, a staining solution, or the like is added to the cells.
Standing: if not specifically stated, the separation is carried out at normal temperature, as long as the cells and the supernatant are well layered, and the separation time can be 10-30 min.
The above operations are carried out at normal temperature unless otherwise specified.
Washing: staining buffer and phosphate buffer containing no calcium or magnesium ions can be used.
Centrifuging: for example, the reaction is carried out under 800-1200G, preferably 800G, and the time can be 5-10 min.
The staining buffer solution is a common physiological buffer solution which contains 0.5-1% (w/v) of blocking protein (such as bovine serum albumin), does not contain calcium and magnesium ions and has a pH range of 7.2-7.4, and preferably is a phosphate buffer solution, such as PBS.
Further, the cells were suspended in the staining buffer after the treatment of step 1). The obtained product can be stored in a refrigerator at-80 deg.C for a long time, such as several months, and can be stored for 1 year at most.
The cells are treated in the steps 1) to 3) and then suspended in a staining buffer, and the cells can be stored in a refrigerator at-80 ℃ for a long time and can be stored for 1 month at most.
Example 1
In this example, the effect of preservation at low temperature (-80 ℃) of erythrocytes isolated from peripheral blood of mice by treatment with glutaraldehyde solution and paraformaldehyde solution was compared.
The specific implementation process is as follows:
1) 5 parts of 0.1ml of fresh mouse (Bal/bc) anticoagulated whole blood was taken, and 0.3ml of PBS (containing no calcium or magnesium ions, unless otherwise specified, the same shall apply hereinafter) was added thereto and mixed well to obtain 0.4ml of single cell suspension.
2) 1.6ml of a glutaraldehyde solution having a concentration of 2%, 2.5%, 3%, 5% (v/v) or 1.6ml of a 4% paraformaldehyde solution was added dropwise thereto, respectively, while continuously vortexing the cell suspension, and allowed to stand at ordinary temperature for 20 min.
3) The cells were washed 2 times with PBS by centrifugation (800G, 5min) and the supernatant was discarded;
4) the treated cells were suspended in 1ml of a staining buffer (1XPBS, HyClone) containing 1% (w/v) of bovine serum albumin (BSA, Sigma) in PBS (1xPBS, HyClone) and not containing calcium and magnesium ions, as not specifically mentioned, hereinafter the same), and frozen in a freezer at-80 ℃;
5) after 2 weeks, the cryopreserved cells were removed, thawed naturally at room temperature, centrifuged (800G, 5min), the supernatant was discarded, the color of the supernatant in the tube was observed, and red blood was observed to indicate that red blood cells were damaged.
The cells treated with a glutaraldehyde solution of 2.5% (v/v) concentration were divided into 2 parts, and the cell morphology was observed by a Scanning Electron Microscope (SEM) and a laser scanning confocal microscope (CLSM), respectively.
A photograph of the supernatant after treatment with glutaraldehyde and paraformaldehyde is shown in FIG. 1 a.
The results show that: the supernatants obtained by treatment with glutaraldehyde solutions at concentrations of 2%, 2.5%, 3%, 5% (v/v) were colorless and transparent, while the supernatants obtained by treatment with paraformaldehyde solutions were red. It is preliminarily presumed that the red blood cells treated with paraformaldehyde solution were damaged and released hemoglobin after the freezing and thawing process, so that the supernatant appeared red (right side of FIG. 1 a), while the red blood cells remained morphologically intact after the treatment with 2.5% (v/v) glutaraldehyde solution (left side of FIG. 1 a).
FIG. 1b shows the morphology of glutaraldehyde-treated erythrocytes observed by SEM, FIG. 1c and FIG. 1d by CLSM. As can be seen from FIGS. 1b, 1c and 1d, after the treatment with the erythrocyte fixative of the present invention containing glutaraldehyde, and the cryopreservation and thawing, the cell membrane of the erythrocytes remains intact, and the whole cell morphology is oval, which can be used for the subsequent single cell detection.
It was further demonstrated that the intact preservation of erythrocytes for a long time can be achieved by using glutaraldehyde solutions of 2%, 2.5%, 3%, 5% (v/v), in particular of 2.5% (v/v), as the erythrocyte fixative. The reason for this is presumed to be that glutaraldehyde solution influences protein configuration by forming intermolecular cross-links, thereby fixing the morphological structure of cells.
Example 2
Suitable concentrations of osmate label for determining the distribution of individual cellular elements are investigated in this example.
1) Blood sampling: preparing 3 female Balb/c mice, and putting whole blood of the mice into a heparin sodium anticoagulation tube;
2) adding 0.1ml of the whole blood into 0.3ml of PBS, and uniformly mixing to obtain 0.4ml of cell suspension;
3) dropwise adding 1.6ml of 2.5% (v/v) glutaraldehyde solution while continuously vortexing the cell suspension, and standing at normal temperature for 20 min;
4) the cells were washed 2 times with PBS by centrifugation (800G, 5min) and the supernatant was discarded;
5) all cells were suspended in 1ml of staining buffer and stored in a freezer at-80 ℃;
6) taking out the frozen cells, naturally thawing at room temperature, centrifuging (800G for 5min), and discarding the supernatant;
7) taking about 4 × 10 obtained by thawing6Suspending the cells in 50. mu.l of staining buffer, adding 50. mu.l of staining buffer containing 1.1. mu.l of anti-mouse Ter119-154Sm antibody (3154005B, Fuluda), mixing well, and standing at room temperature for 30 min;
8) cells were washed 2 times by centrifugation (800G, 5min) with staining buffer, and the supernatant was discarded to contain about 4X 10 per 100. mu.l6The density of individual cells was dispersed in PBS;
9) to 100. mu.l of each of the above cell PBS suspensions was added dropwise 1ml of 5X 10-6％、10-5％、3×10-5％、5×10-5％、7×10-5％、10-4％、5×10-5% w/v osmate solution while vortexing the cell suspension continuously and standing at room temperature for 10 min;
10) cells were washed 2 times by centrifugation (800G, 5min) with staining buffer and the supernatant was discarded;
11) suspending the cells in 1ml of staining buffer, and storing in a refrigerator at-80 ℃;
12) before detecting lead element in single erythrocyte by using mass flow cytometry (Helios), the cell frozen at-80 ℃ is naturally thawed, and the cell is washed by using ultrapure water for 3 times by centrifugation (800G, 5min) and then loaded. EQbeads were used for internal reference.
Mass cytometry detection parameters:
cell suspension sample injection flow rate: 30 to 31 μ l/min,
the rate of event collection is < 500 events/s,
EQbeads concentration: 10 to 15 percent of the total weight of the mixture,
EQbeads monitoring element: 140/142Ce (cerium), 151/153Eu (europium), 165Ho (holmium), 175/176Lu (lutetium);
elements to be tested:154Sm(Ter119)、78Se、88Sr、127I、192Os、190Os、208Pb、209Bi。
the results of the detection are shown in FIG. 2.
As a result: in fig. 2a, the Y-axis is the proportion of individual cells labelled with 7 different concentrations of osmate and the X-axis is the osmate concentration. Among them, 5X 10 is known-6% osmate concentration, the proportion of stained single cells was significantly lower than other concentrations, confirming that this concentration stained poorly and was not suitable as a condition for single cell detection.
FIGS. 2 b-2 h are scattergrams of a flow cytometer, where each dot represents a cell, and dark represents high cell density and light represents low cell density. Scatter plots further confirm that osmic acid concentration is 5X 10-6(w/v), the cell population was dispersed, confirming that the staining effect at this concentration was poor; in osmic acid solution at concentration of 5X 10-4In% (w/v), the upper end of the cell population was clearly shifted to the right, suggesting that the staining signal was too strong, affecting the distribution and identification of the individual cell population, confirming that the concentration effect was not suitable for the detection of the individual cells.
In conclusion, the osmic acid working concentration in the staining solution of the invention is 10-5％～10-4In the range of% (w/v), when the antibody is used in combination with a metal-labeled antibody specific to erythrocytes as the metal-labeled antibody, good staining and detection effects can be obtained. Wherein the concentration is 5 × 10-5In% (w/v), the distribution of individual cells was concentrated, and it was clearly seen that the osmic acid signal was approximately for the right cell population (shown as circles) representing 2 cell populationsThe left 2-fold indicates that the right cell population is cell dimers, which is more preferable because it distinguishes between single cells and dimers and multicellular aggregates.
Example 3
The stability over time of erythrocytes treated with the erythrocyte fixative.
1) Taking 0.1ml of fresh human anticoagulated whole blood, adding the anticoagulated whole blood into PBS with the volume of 0.3ml, and uniformly mixing to obtain 0.4ml of cell suspension;
2) dropwise adding 1.6ml of 2.5% (v/v) glutaraldehyde solution while continuously vortexing the cell suspension, and standing at normal temperature for 20 min;
3) the cells were washed 2 times with PBS by centrifugation (800G, 5min) and the supernatant was discarded;
4) suspending all cells in 1ml of staining buffer solution, dividing into 2 parts, and storing in a refrigerator at minus 80 ℃ for later use;
5) taking out a frozen cell when the preservation time is 15 days and 150 days respectively, naturally thawing at room temperature, centrifuging (800G, 5min), and discarding the supernatant for subsequent labeling;
6) collecting and recovering to obtain about 4 × 106Suspending the cells in 50 μ l staining buffer, adding 50 μ l staining buffer containing 1.1 μ l anti-human CD235ab-141Pr antibody (3141001B, Fuluda), mixing, and standing at room temperature for 30 min;
7) cells were washed 2 times by centrifugation (800G, 5min) with staining buffer, and the supernatant was discarded to contain about 4X 10 per 100. mu.l6The density of individual cells was dispersed in PBS;
8) to 100. mu.l of the above cell PBS suspension was added dropwise 1ml of 5X 10-5% w/v osmate solution while vortexing the cell suspension continuously and standing at room temperature for 10 min;
9) cells were washed 2 times by centrifugation (800G, 5min) with staining buffer and the supernatant was discarded;
10) suspending the cells in 1ml of staining buffer, and storing in a refrigerator at-80 ℃;
11) after 24h, the cells frozen at-80 ℃ were thawed naturally, washed 3 times with ultrapure water by centrifugation (800G, 5min) and loaded, and single-cell detection was performed using a mass-flow cytometer (Helios)Praseodymium in individual red blood cells141Pr (CD235ab), selenium78Se, Sr88Sr and iodine127I. Osmium (III)192Os, Os190Os, lead208Pb and Bi 209Bi elements. EQbeads were used for internal reference.
Mass cytometry detection parameters:
cell suspension sample injection flow rate: 30 to 31 μ l/min,
the rate of event collection is < 500 events/s,
EQbeads concentration: 10 to 15 percent of the total weight of the mixture,
EQbeads monitoring element: 140/142Ce (cerium), 151/153Eu (europium), 165Ho (holmium), 175/176Lu (lutetium);
elements to be tested: praseodymium (III)141Pr (CD235ab), selenium78Se, Sr88Sr and iodine127I. Osmium (III)192Os, Os190Os, lead208Pb and Bi209Bi。
The detection results are shown in FIGS. 3(a) to (c).
As a result: the two-dimensional flow type scatter diagram shows that the distribution of single cell groups stored for 15 days and 150 days at-80 ℃ is similar and has no obvious difference, and the single cell proportion of the single cell groups is very close to that of the single cell groups, so that the red blood cell sample stored for a long time is not changed in the storage process, and the kit can be used for storing the red blood cell sample for a long time.
FIGS. 3(d) to (h) show elements, respectively78Se、88Sr、127I、208Pb、209And (b) a single-cell distribution diagram of Bi, wherein each dot in the diagram represents one cell, and the value of the content of the element in each single cell is expressed in terms of color as shown by the color bar on the right side of each diagram, the closer the color is to red at the upper end of the color bar, the higher the content of the element in the dot, the closer the color is to deep blue at the lower end of the color bar, and the lower the content of the element in the dot. The results show that: there are a few deep red (very high), orange (high) spots and most dark blue (very low) spots in praseodymium, selenium, strontium, iodine, bismuth, suggesting that the distribution pattern of these elements is such that high content of elements is concentrated in a few red blood cells in the sample, whereas for the case of praseodymium, selenium, strontium, iodine, bismuth208Pb element, mostlyThe dots are light blue (lower) to light yellow (middle), which indicates that in the sample, the distribution mode of the lead element is that the low-middle content element is distributed in a large number of red blood cells, and has difference with other elements, and the content of the lead element is higher than that of other elements in whole, and the average amount is between 7 and 25counts (signal intensity).
Example 4
The staining solution kit of the present invention detects lead distribution of individual cells among mouse spleen leukocytes (nucleated cells).
Female Balb/c mice were 6, divided into lead nitrate group (3) and control group (3). Injection of 200. mu.l PBS (control group) or 200. mu.l 5mM lead nitrate solution (exposed group) from tail vein, respectively;
after 4h of injection, dissecting a mouse, taking the spleen, preparing a spleen single cell suspension, and cracking erythrocytes by using an erythrocyte lysate (erythrocyte lysate, Solibao) to obtain a leukocyte suspension;
the leukocyte suspension was divided into two equal parts, and labeled and detected using the kit and method of the present invention (hereinafter referred to as osmium labeling method), respectively. That is, the following tests were performed by dividing the samples into four samples, i.e., a control group + osmium labeling method, an exposed group + osmium labeling method, a control group + iridium labeling method, and an exposed group + iridium labeling method:
control group + osmium labeling method, exposure group + osmium labeling method
1) Taking 0.4ml of leukocyte suspension, dropwise adding 1.6ml of 2.5% (v/v) glutaraldehyde solution into the leukocyte suspension, continuously swirling the cell suspension, and standing at normal temperature for 20 min;
2) the cells were washed 2 times with PBS by centrifugation (800G, 5min) and the supernatant was discarded;
3) take 4X 106Suspending the cells in 50 μ l staining buffer, adding 50 μ l staining buffer containing 1.1 μ l anti-mouse CD45-147Sm antibody (3147003B, Fuluda), mixing, and standing at room temperature for 30 min;
4) cells were washed 2 times with staining buffer by centrifugation (800G, 5min), and the supernatant was discarded to contain about 4X 10 per 100. mu.l6The density of individual cells was dispersed in PBS;
5) to 100. mu.l of the above cell PBS suspension was added dropwise1ml of 5X 10-5% w/v osmate solution while vortexing the cell suspension continuously and standing at room temperature for 10 min;
6) cells were washed 2 times by centrifugation (800G, 5min) with staining buffer and the supernatant was discarded;
7) washing the cells with ultrapure water by centrifugation (800G, 5min) for 3 times and loading; and detecting the lead element by using a mass cytometry. EQbeads were used for internal reference.
Mass spectrometry flow cytometer (Helios) detection parameters
Cell suspension sample injection flow rate: 30 to 31 μ l/min,
the rate of event collection is < 500 events/s,
EQbeads concentration: 10 to 15 percent of the total weight of the mixture,
EQbeads monitoring element: 140/142Ce (cerium), 151/153Eu (europium), 165Ho (holmium), 175/176Lu (lutetium); the elements to be tested include:154samarium (Ter119) and osmium192Os, Os190Os, lead208Pb。
Control group + iridium labeling method, exposure group + iridium labeling method
1) Take 4X 106Suspending the cells in 50 μ l staining buffer, adding 50 μ l staining buffer containing 1.1 μ l anti-mouse CD45-147Sm antibody (3147003B, Fuluda), mixing, and standing at room temperature for 30 min;
2) cells were washed 2 times with staining buffer by centrifugation (800G, 5min), and the supernatant was discarded to contain about 4X 10 per 100. mu.l6The density of individual cells was dispersed in PBS;
3) iridium labeling was performed according to the instructions of the commercial kit (201192B, FLUIDIGM);
4) detecting lead element by using a mass cytometry; the internal reference was Eqbeads.
Mass spectrometry flow cytometer (Helios) detection parameters
Cell suspension sample injection flow rate: 30 to 31 μ l/min,
the rate of event collection is < 500 events/s,
EQbeads concentration: 10 to 15 percent of the total weight of the mixture,
EQbeads monitoring element: 140/142Ce (cerium), 151/153Eu (europium), 165Ho (holmium)) 175/176Lu (lutetium); the elements to be tested include:154samarium (Ter119), Iridium191Ir, Ir193Ir, lead208Pb。
The results of the measurement are shown in FIG. 4, and FIG. 4(a) shows the ratio of single cells in leukocytes: in the white blood cells of the control group, the single cell ratio obtained by adopting the osmium labeling method is higher than that obtained by adopting the iridium labeling method, and in the exposed group, the single cell ratio obtained by adopting the osmium labeling method is also higher than that obtained by adopting the iridium labeling method.
FIG. 4(b) is a proportion of lead-containing single cells in leukocytes. It can be seen that in the control group which is not exposed to exogenous lead, the single cells containing lead in the mouse white blood cells can be detected more sensitively by using the osmium labeling method; in the exposed group, the ratio of lead-containing single cells in the white blood cells obtained by the osmium labeling method was similar to that obtained by the iridium labeling method, suggesting that the osmium labeling method is an effective method for labeling white blood cells in addition to labeling red blood cells.
The above results illustrate that: the kit and the detection method thereof can also be used for marking the nucleated cells, and in addition, the kit and the detection method thereof have the marking effect which is equivalent to that of the commercialized iridium element.
The purpose, technical solution and advantages of the present invention are further described in detail with reference to the embodiments, it should be understood that the above-mentioned embodiments are only examples of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Industrial applicability
Compared with the traditional total amount detection technology, the staining solution, the kit and the detection method using the staining solution and the kit solve the problem of detection of average element content in single cells, particularly red blood cells, and by simultaneously using various antibodies, dozens of element information in single red blood cells are expected to be simultaneously detected, so that technical support is provided for scientific research purposes and environmental pollution monitoring. The staining solution, the kit and the detection method using the staining solution and the kit can realize the measurement of various indexes of red blood cells after long-term low-temperature preservation by combining the staining solution, the metal labeled antibody and the red blood cell preservation solution, and can be widely applied to the purposes of scientific research, environment protection of heavy metal pollution, the field of monitoring, biological sample detection, drug development and the like.
Claims (11)
1. A staining solution for labeling cells (e.g., erythrocytes or leukocytes) in mass cytometry comprising osmate at a working concentration of the staining solution of 10-5％～10-4% (w/v), preferably 3X 10-5％～7×10-5% (w/v), more preferably 5X 10-5％～7×10-5％(w/v)。
2. A kit comprising the staining solution of claim 1, further comprising: the metal-labeled antibody is preferably a cell-specific metal-labeled antibody.
3. The kit of claim 1 or 2, wherein: when the cells are red blood cells, the kit further comprises a red blood cell shaping solution, and the red blood cell shaping solution contains glutaraldehyde with the concentration of 2-5% (v/v), preferably 2-3% (v/v).
4. The kit according to any one of claims 2 to 3, wherein the cell-specific metal-labeled antibody is a lanthanide-labeled antibody (preferably one or more of an anti-mouse Ter 119-lanthanide-labeled antibody, an anti-human CD235 ab-lanthanide-labeled antibody, an anti-mouse CD 45-lanthanide-labeled antibody, or an anti-human CD 45-lanthanide-labeled antibody), optionally wherein the lanthanides in the lanthanide-labeled antibodies used together are different from each other at the time of detection.
5. The kit according to claim 4, wherein the lanthanide in the lanthanide-labelled antibody is selected from La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), scandium (Sc), yttrium (Y), preferably Sm (samarium), Pr (praseodymium).
6. The kit according to any one of claims 2 to 5, further comprising a buffer for staining, which is a buffer comprising 0.5% to 1% (w/v) of a blocking protein (such as bovine serum albumin) and being free of calcium and magnesium ions (preferably a phosphate buffer, more preferably PBS, having a pH range of 7.2 to 7.4).
7. The kit according to claims 2 to 6, further comprising a cell wash solution, wherein the cell wash solution (preferably phosphate buffer solution with pH ranging from 7.2 to 7.4, more preferably PBS) does not contain calcium and magnesium ions, preferably the concentration of phosphate buffer solution without the magnesium ions is 0.0067M.
8. Use of a kit according to any one of claims 2 to 7 in the detection of individual cells, wherein preferably the cells to be detected comprise red blood cells.
9. Application of glutaraldehyde solution with concentration of 2-5% (v/v) in long-term preservation of erythrocytes.
10. A method of single-cell elemental detection comprising the steps of:
treating the cells with a glutaraldehyde solution at a concentration of 2% to 5% (v/v), preferably 2% to 3% (v/v);
using osmic acid in a concentration of 10-5～10-4% (w/v), preferably 3X 10-5～7×10-5% (w/v), more preferably 5X 10-5～7×10-5% w/v of the staining solution for labeling the cells (preferably, the concentration of the cells is 2X 106～4×106One/ml);
the cells are labeled with a metal-labeled antibody, preferably a cell-specific metal-labeled antibody.
11. The method of claim 10, further comprising one or more of the following steps:
dissolving the metal-labeled antibody in a staining buffer as defined in claim 6;
washing the cells with a staining buffer as defined in claim 6;
washing the cells using a cell wash as defined in claim 7.
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| CN111982789A (en) * | 2020-08-21 | 2020-11-24 | 中国科学院生态环境研究中心 | High-throughput detection method of metal ions and metal nanoparticles based on single-cell enrichment and single-cell mass spectrometry |
| WO2021139420A1 (en) * | 2020-01-06 | 2021-07-15 | 中国科学院生态环境研究中心 | Test kit used for single-cell tagging of red blood cells and detection method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3776697A (en) * | 1972-04-13 | 1973-12-04 | Atomic Energy Commission | Method and reagent for determining nitrogen oxides |
| SU1427238A1 (en) * | 1985-05-29 | 1988-09-30 | Предприятие П/Я А-7345 | Electronic-optical method of determining molecular masses and molecular-mass distribution of butadiene polymers |
| CN101379387A (en) * | 2005-12-30 | 2009-03-04 | 霍尼韦尔国际公司 | Differential white blood count on a disposable card |
| CN102335434A (en) * | 2010-07-23 | 2012-02-01 | 复旦大学 | Targeting carbon nanotube drug carrier with specific monoclonal antibody and its preparation method |
| CN104075981A (en) * | 2013-03-29 | 2014-10-01 | 希森美康株式会社 | Blood cell analyzer and blood cell analyzing method |
| CN108398361A (en) * | 2018-05-25 | 2018-08-14 | 西湖大学 | The method and its application lapsed to using red blood cell DNA damage signal estimation blood disease |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103900883B (en) * | 2014-04-10 | 2016-08-31 | 甘肃农业大学 | Cashmere goat skin is for the preparation method of the ultrathin section of transmission electron microscope observing |
| CN104792806B (en) * | 2015-04-22 | 2017-07-04 | 山东省农业科学院作物研究所 | A kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method |
| CN106053178B (en) * | 2016-07-28 | 2018-08-31 | 广西医科大学 | A kind of preparation method of sterculia seed scanning electron microscope example |
| WO2018073283A1 (en) * | 2016-10-19 | 2018-04-26 | F. Hoffmann-La Roche Ag | Systems and methods for staining of biological samples |
| CN111157433A (en) * | 2020-01-06 | 2020-05-15 | 中国科学院生态环境研究中心 | Kit for marking single cell of red blood cell and detection method thereof |
-
2020
- 2020-01-06 CN CN202010009183.9A patent/CN111157433A/en active Pending
- 2020-11-23 WO PCT/CN2020/130805 patent/WO2021139420A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3776697A (en) * | 1972-04-13 | 1973-12-04 | Atomic Energy Commission | Method and reagent for determining nitrogen oxides |
| SU1427238A1 (en) * | 1985-05-29 | 1988-09-30 | Предприятие П/Я А-7345 | Electronic-optical method of determining molecular masses and molecular-mass distribution of butadiene polymers |
| CN101379387A (en) * | 2005-12-30 | 2009-03-04 | 霍尼韦尔国际公司 | Differential white blood count on a disposable card |
| CN102335434A (en) * | 2010-07-23 | 2012-02-01 | 复旦大学 | Targeting carbon nanotube drug carrier with specific monoclonal antibody and its preparation method |
| CN104075981A (en) * | 2013-03-29 | 2014-10-01 | 希森美康株式会社 | Blood cell analyzer and blood cell analyzing method |
| CN108398361A (en) * | 2018-05-25 | 2018-08-14 | 西湖大学 | The method and its application lapsed to using red blood cell DNA damage signal estimation blood disease |
Non-Patent Citations (4)
| Title |
|---|
| HIDEKI MAKINOSHIMA,ET AL.: "Growth Phase-Coupled Alterations in Cell Structure and Function of Escherichia coli", 《JOURNAL OF BACTERIOLOGY》 * |
| RAUL CATENA,ET AL.: "Enhanced Multiplexing in Mass Cytometry Using Osmium and Ruthenium Tetroxide Species", 《CYTOMETRY PART A》 * |
| 李兴程 等: "维生素E对CBA小鼠年龄相关性听力损失的保护作用及其机制", 《临床与病理杂志》 * |
| 杨贵贞: "《医用免疫学》", 31 July 1980, 吉林人民出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021139420A1 (en) * | 2020-01-06 | 2021-07-15 | 中国科学院生态环境研究中心 | Test kit used for single-cell tagging of red blood cells and detection method thereof |
| CN111982789A (en) * | 2020-08-21 | 2020-11-24 | 中国科学院生态环境研究中心 | High-throughput detection method of metal ions and metal nanoparticles based on single-cell enrichment and single-cell mass spectrometry |
| CN111982789B (en) * | 2020-08-21 | 2022-02-08 | 中国科学院生态环境研究中心 | High-throughput detection method of metal ions and metal nanoparticles based on single-cell enrichment and single-cell mass spectrometry |
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