WO2021139420A1

WO2021139420A1 - Test kit used for single-cell tagging of red blood cells and detection method thereof

Info

Publication number: WO2021139420A1
Application number: PCT/CN2020/130805
Authority: WO
Inventors: 胡立刚; 刘念; 王丁一; 曲广波; 何滨; 江桂斌
Original assignee: 中国科学院生态环境研究中心
Priority date: 2020-01-06
Filing date: 2020-11-23
Publication date: 2021-07-15
Also published as: CN111157433A

Abstract

A staining liquid used for tagging cells in mass spectrum flow cytometry, a test kit and detection method including said staining liquid, and a red blood cell fixative liquid. The staining liquid containing osmic acid, and the test kit further comprising a cell-specific metal-tagged antibody. By means of using the test kit together with the red blood cell fixative liquid, the test kit is particularly applicable for tagging and detection of individual red blood cells, and may be used for detection of distribution among individual red blood cells of multiple elements.

Description

Reagent kit for single cell labeling of red blood cells and detection method thereof

Technical field

The invention relates to a staining solution for cell labeling in mass spectrometry flow 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 the determination of the distribution of multiple elements in a single cell of non-nucleated cells, especially red blood cells.

Background technique

Mass flow cytometry (cytometry by time-of-flight, CyTOF) is a single-cell detection technology that has developed very rapidly in recent years. It is a flow cytometer based on the principle of mass spectrometry, which uses the metal element iridium (Ir) or rhodium. (Rh) labeling cell nucleus for screening and identification of single cells; using non-radioactive rare earth elements as antibody labels, this method has accurate signals, multiple detection channels, and high stability. Because there is no need to calibrate the overlapping interference of the spectrum, it is compatible with traditional mass spectrometers. In contrast, this method can simultaneously detect the information of up to 135 elements in a single cell, which is an effective research method.

Red blood cells are the main cell type of blood cells. They not only have the function of transporting oxygen and carbon dioxide, but also have the functions of immune adhesion and immune phagocytosis. They also play a buffering role in the acid-base balance and are very important for maintaining the normal physiological activities of the living body. .

The element content in red blood cells reflects the bioaccumulation of elements and the law of their transfer and transformation in the human body, and is closely related to human health. There is individual heterogeneity in the distribution of element content among individual red blood cells. Some red blood cells have high element content and some red blood cells have low element content. The element content is closely related to its function and health effects. Analyzing the element content in a single red blood cell is of great significance to the study of its distribution law, metastasis and transformation law, and related physiology and pathology.

At present, the detection of element content in red blood cells is limited to total detection. The detection methods mainly include ultraviolet visible spectrophotometry (UV), atomic absorption method (AAS), inductively coupled plasma emission spectrometer (ICP-OES), and inductively coupled plasma Mass spectrometry (ICP-MS) and so on. 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 performing single-cell detection of red blood cells, the main obstacle is that red blood cells do not have a nucleus, so they cannot be labeled by the metal elements iridium (Ir) or rhodium (Rh) currently used in mass spectrometry flow cytometry; in addition, red blood cells One of the major characteristics of the cell is that it is easy to rupture, and it is very easy to rupture due to changes in the osmotic pressure inside and outside the cell, so it is difficult to ensure the integrity of the cell shape during the detection process.

Moreover, in practice, it is often difficult to perform mass spectrometry flow cytometry measurements at the first time after blood collection, depending on the sample collection conditions, the arrangement of test equipment, and the scheduling of parallel tests. Therefore, it is desirable to have a reagent and method that can keep the red blood cell sample intact and preserve it for a long time.

In view of the above reasons, there is a need for new technical support for the detection of elements in a single red blood cell, as well as long-term preservation agents that can be used for scientific research and commercial use to keep the red blood cells intact.

Summary of the invention

Aiming at the deficiencies of the prior art, the present invention provides a staining solution, a kit and a detection method suitable for single cell element detection of non-nucleated cells, especially red blood cells. The kit can realize long-term preservation of red blood cells in vitro, and is suitable for single cell element detection by mass spectrometry flow cytometer for red blood cells.

Osmium acid is a lipid-reactive agent. When using transmission electron microscopy to observe tissue cells, osmium acid fixative can be used to stain cell membranes well, which can be used for the preparation of transmission electron microscopy pathological diagnosis samples. In such applications, the concentration of osmium acid used is generally 2% (w/w), and sometimes 1% (w/w) is used.

In flow cytometry, the labeling amount of elemental osmium is strictly related to the size of the labeled cells. Therefore, the signal of elemental osmium can be used to distinguish between intact single cells and cell fragments to realize the detection of single cells. The inventor found that the working concentration is 10 ^-5 % to 10 ^-4 % (w/v), preferably 3×10 ^-5 % to 7×10 ^-5 % (w/v), more preferably When 5×10 ^-5 %～7×10 ^-5 % (w/v) osmium acid solution is used as a staining solution, it can obtain good results for cell samples, especially red blood cells, in mass spectrometry flow cytometry at the individual cell level. Dyeing effect.

Mass spectrometry flow cytometry replaces traditional fluorescent proteins with lanthanide metals, which are extremely low in cells, to label antibodies. In this context, such antibodies labeled with lanthanides are sometimes referred to as metal-labeled antibodies, in which metal-labeled antibodies that bind to specific cells (hereinafter referred to as cell-specific metal-labeled antibodies) can specifically bind to specific cells. In order to realize the screening and identification of specific cells, for example, red blood cell-specific metal-labeled antibodies can identify red blood cells and distinguish red blood cells from other cell types. In the detection, the lanthanides in the commonly used lanthanide-labeled antibodies are different from each other, and more than 30 or even 40 kinds of antibodies can be used for labeling at the same time. In this article, the capital letter M is sometimes used to represent lanthanides.

The staining solution of the present invention can be used in combination with a metal-labeled antibody to well identify target cells and label single cells, thereby realizing single-cell-level detection for target cells. It should be noted that the staining solution for cell element detection of the present invention can also be used in combination with other cell identification markers commonly used in the art, as long as its function of distinguishing intact cells from cell debris can be used to successfully mark the level of individual cells.

As metal-labeled antibodies, cell-specific metal-labeled antibodies, especially red blood cell-specific metal-labeled antibodies, are used in the present invention. The red blood cell-specific metal-labeled antibodies include, but are not limited to: anti-mouse Ter119-lanthanide-labeled antibodies, At least one of an anti-human CD235ab-lanthanide-labeled antibody, an anti-mouse CD45-lanthanide-labeled antibody, or an anti-human CD45-lanthanide-labeled antibody.

The lanthanides used in the present invention can be, for example, La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), One or more of Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), scandium (Sc), yttrium (Y) It should be noted that during detection, the lanthanides in the lanthanide-labeled antibodies used in common need to be different from each other.

The present invention also provides a red blood cell styling solution that can store red blood cells in vitro, preferably at low temperature, more preferably at -80°C, for long-term storage, which contains a concentration of 2% to 5% (v/v), preferably 2 % To 3% (v/v), particularly preferably 2.5% (v/v) glutaraldehyde solution.

The "long-term preservation in vitro" in the present invention means that after the preservation is completed, the preserved cells can be observed with a scanning electron microscope or other techniques in the field to observe the complete cell morphology, which can be used for the next step of cell biochemical indicators The state of such a measurement is sufficient, and the cell does not have to be a living cell.

The "single cell detection" of the present invention includes simultaneous or batch detection of one or more indicators of a cell population, so as to obtain multiple or one type of single (individual) cells in the cell population. Element content, biochemistry, immunity and various information.

Specifically, the present invention relates to the following:

1. A staining solution for labeling cells (such as red blood cells or white blood cells) in mass spectrometry flow cytometry, which contains osmium acid, and the concentration of the osmium acid is 10 ^-5 % to 10 at the working concentration of the staining solution ^-4 % (w/v), preferably 3×10 ^-5 % to 7×10 ^-5 % (w/v), more preferably 5×10 ^-5 % to 7×10 ^-5 % (w/v ).

2. A kit comprising the staining solution described in item 1, which further comprises: a metal-labeled antibody, 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 sizing solution, and the red blood cell sizing solution contains a concentration of 2% to 5% (v/v ), preferably 2% to 3% (v/v) glutaraldehyde.

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 an anti-mouse Ter119-lanthanide-labeled antibody, an anti-human CD235ab -One or more of lanthanide-labeled antibody, anti-mouse CD45-lanthanide-labeled antibody or anti-human CD45-lanthanide-labeled antibody), optionally, the lanthanide that is commonly used in the detection The lanthanides in the labeled antibodies are different from each other.

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 (Eu), Gd (Gadolinium), Tb (Tb), Dy (Dy (Dy), Ho (Ho), Er (Erbium), Tm (Thulium), Yb (Ytterbium), Lu (Lute ), scandium (Sc), yttrium (Y), preferably Sm (samarium), Pr (praseodymium).

6. The kit according to any one of items 2 to 5, further comprising a staining buffer, the staining buffer containing 0.5% to 1% (w/v) of blocked protein (such as bovine serum Albumin) and a buffer containing no calcium and magnesium ions (preferably a phosphate buffer with a pH range of 7.2 to 7.4, more preferably PBS).

7. The kit according to items 2 to 6, further comprising a cell washing solution (preferably a phosphate buffer with a pH range of 7.2 to 7.4, more preferably PBS) containing no calcium and magnesium Ions, preferably, the concentration of the phosphate buffer without the magnesium ions is 0.0067M.

8. The use of the kit according to any one of items 2 to 7 in the detection of a single cell, wherein the cells to be detected preferably include red blood cells.

9. A method for single cell element detection, including the following steps:

The utilization concentration is 2%～5%(

v/v), preferably 2% to 3% (v/v) glutaraldehyde solution to treat cells;

The concentration of osmic acid used is 10 ^-5 ～10 ^-4% (w/v), preferably 3×10 ^-5 ～7×10 ^-5 % (w/v), more preferably 5×10 ^-5 ～7 ×10 ^-5 % (w/v) staining solution to label cells (wherein, the concentration of cells is preferably 2×10 ⁶ to 4×10 ⁶ cells/ml);

The cells are labeled with a metal-labeled antibody (preferably a cell-specific metal-labeled antibody).

10. The method according to item 9, further comprising one or more of the following steps:

Dissolving the metal-labeled antibody in a staining buffer, the staining buffer being a buffer containing 0.5% to 1% (w/v) blocked protein and free of calcium and magnesium ions;

Washing the cells with the staining buffer;

Wash the cells with a cell wash, which is a phosphate buffer without calcium and magnesium ions

The staining solution, kit and detection method provided by the present invention are particularly suitable for the detection of single cell elements of non-nucleated cells, especially red blood cells, by using osmium acid solution as a staining solution, glutaraldehyde solution as a red blood cell setting solution, and red blood cells Specific metal-labeled antibodies can achieve morphological fixation of red blood cells, specific screening and red blood cell membrane labeling.

It should be noted that although osmium tetroxide solution is often used as a fixative in the field of cell detection, the concentration of osmium tetroxide in the solution used in the present invention is much lower than the concentration of osmium tetroxide used for cell fixation. Those are not in the same order of magnitude, and the nature of the osmium used is also different.

Description of the drawings

Fig. 1 is a graph showing the integrity of red blood cells treated with the red blood cell sizing solution of the present invention. among them,

Figure (a) shows the situation after treatment with glutaraldehyde and paraformaldehyde (PFA) solutions, the left is the glutaraldehyde solution, and the right is the paraformaldehyde solution;

Figure (b) is the scanning electron microscope morphology of red blood cells fixed with glutaraldehyde solution;

Figure (c) is the bright field morphology of red blood cells fixed with glutaraldehyde solution under a laser scanning confocal microscope;

Figure (d) is a graph of the fluorescence (Ter119-PE) morphology of red blood cells fixed in glutaraldehyde solution and observed with a laser scanning confocal microscope.

Figure 2 shows the labeling effect of different concentrations of osmium acid, where (a) is the proportion of individual cells detected by mass spectrometry flow cytometry after labeling with different concentrations of osmium acid; 5×10 ^-6 %, 10 ^-5 %, 3×10 ^-5 %, 5×10 ^-5 %, 7×10 ^-5 %, 10 ^-4 %, 5×10 ^-5 % (w/v) A two-dimensional scatter plot of a single cell labeled with osmium acid.

Figure 3 shows the red blood cell sample treated with the red blood cell sizing solution, where (a) is the proportion of single cells in the sample stored for 15 days and 150 days after the red blood cell sizing solution; (b) and (c) are stored 15 days Two-dimensional scatter plots of single cells at day and 150 days, (d)-(h) are ^{the distribution maps of elements 78} Se, ⁸⁸ Sr, ¹²⁷ I, ²⁰⁸ Pb, and ²⁰⁹ Bi in a single cell, respectively.

Figure 4 is a comparison diagram of nucleated cell detection results obtained by using the kit and method of the present invention and the iridium element labeling method. Among them, (a) is a diagram of the proportion of white blood cells labeled with iridium element and the kit of the present invention; (b) is the lead-containing white blood cells (exposed group) after exposure to lead is obtained using the iridium element label and the kit of the present invention. Cell scale diagram.

Detailed ways

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments with reference to the accompanying drawings.

As an aspect of the present invention, there is provided an application of glutaraldehyde solution in long-term preservation of red blood cells, and the method of using the same for preserving red blood cells includes the following steps, for example:

■ Take fresh human or animal anticoagulated whole blood, add 3 times its volume of phosphate buffer (without calcium and magnesium ions), mix well to obtain a single cell suspension;

■ Add dropwise a glutaraldehyde solution with a concentration of 2% to 5% (v/v) that is 4 times the volume of the cell suspension, and let it stand at room temperature for 10 minutes to 30 minutes;

■ Wash the cells with phosphate buffer (without calcium and magnesium ions) by centrifugation;

■ Cell cryopreservation: Suspend cells in a buffer containing 1 to 3% (w/v) blocked protein (such as bovine serum albumin) and without calcium and magnesium ions, or commonly used in the field for cell cryopreservation The solution is frozen and stored in a refrigerator at -80℃.

As another aspect of the present invention, there is provided a single cell detection kit and a method of using the same, the method comprising the following steps:

■ Add red blood cell sizing solution dropwise to the cell suspension, and let it stand at room temperature for 10-30 minutes;

■ Take 2×10 ⁶ ～4×10 ⁶ cells of each sample and suspend them in staining buffer, add an equal volume of staining buffer containing metal-labeled antibodies (wherein, the dilution factor of the antibody is in accordance with the instructions), and mix well. Let stand at room temperature for 30 minutes, and then wash the cells by centrifugation with staining buffer;

It should be noted that although 2×10 ⁶ to 4×10 ⁶ cells are used here, the effects of the present invention can also be achieved within the range of cell concentration of 1×10 ⁶ to 8×10 ^{6 cells/ml} ；

■ Labeling with osmic acid solution: Suspend all the above cells in 100μl staining buffer, add 1ml ^{10 -5} ～10 ^-4 % (w/v) osmium acid solution, while continuing to vortex the cell suspension, and let it stand at room temperature for 10 min～ 30min, then wash the cells by centrifugation with staining buffer;

■ After washing the cells with ultrapure water 3 times by centrifugation, the mass spectrometry flow cytometer is used to complete the detection of elements in a single red blood cell.

Red blood cell styling solution is a reagent that can fix red blood cells without rupturing red blood cells, preferably at a concentration of 2% to 5% (v/v), more preferably 2% to 3% (v/v) of glutaraldehyde Solution.

Furthermore, the staining solution, the kit, and the red blood cell sizing solution of the present invention are preferably stored at a low temperature, and the storage conditions are, for example, 2 to 8°C.

Dispersion: It is preferable to perform dispersion by vortexing or pipetting when adding red blood cell sizing solution, staining solution, etc. to the cells.

Let stand: if there is no special instructions, all are carried out at room temperature, as long as the cells and the supernatant reach a good stratification, it can be 10-30min.

The above operations are carried out at room temperature unless otherwise specified.

Washing: Able to use staining buffer and phosphate buffer without calcium and magnesium ions.

Centrifugation: for example, 800-1200G, preferably 800G, the time can be 5-10min.

The staining buffer of the present invention is a common physiological buffer containing 0.5 to 1% (w/v) of occluded protein (such as bovine serum albumin) and containing no calcium and magnesium ions in the pH range of 7.2 to 7.4, preferably The commonly used physiological buffer is phosphate buffer, such as PBS.

Further, the cells are suspended in the staining buffer after being processed in the step 1). In this state, it can be stored in a -80°C refrigerator for a long time, for example, several months, and can be stored for up to 1 year.

After the cells are processed in steps 1)-3), they are suspended in a staining buffer. In this state, they can be stored in a refrigerator at -80°C for a long time, and can be stored for a maximum of 1 month.

Example 1

In this example, the low-temperature (-80°C) preservation effect of glutaraldehyde solution and paraformaldehyde solution treatment on red blood cells isolated from peripheral blood of mice was compared.

The specific implementation process is as follows:

1) Take 5 0.1ml fresh mouse (Bal/bc) anticoagulated whole blood, add 0.3ml PBS (without calcium and magnesium ions, unless otherwise specified, the following are the same), mix well to obtain 0.4ml single cell Suspension.

2) Add 1.6ml of 2%, 2.5%, 3%, 5% (v/v) glutaraldehyde solution or 1.6ml 4% paraformaldehyde solution into it drop by drop, while continuing to vortex the cell suspension, Let stand for 20min at room temperature.

3) Wash the cells twice with PBS by centrifugation (800G, 5min), and discard the supernatant;

4) Suspend the treated cells in 1ml staining buffer (containing 1% (w/v) of bovine serum albumin (BSA, Sigma) in PBS (1xPBS, HyClone), without calcium and magnesium ions, such as No special instructions, the following are the same), frozen in a refrigerator at -80℃;

5) Take out the frozen cells after 2 weeks, thawed naturally at room temperature, centrifuge (800G, 5min), discard the supernatant, observe the color of the supernatant in the tube, red blood cells are considered to be damaged.

The cells treated with 2.5% (v/v) glutaraldehyde solution were divided into two parts, and the cell morphology was observed by scanning electron microscope (SEM) and laser scanning confocal microscope (CLSM) respectively.

The photo of the supernatant treated with glutaraldehyde and paraformaldehyde is shown in Figure 1a.

The results showed that the supernatant obtained by treatment with glutaraldehyde solutions with concentrations of 2%, 2.5%, 3%, and 5% (v/v) was colorless and transparent, while the supernatant treated with the paraformaldehyde solution was red. It can be preliminarily inferred that the red blood cells treated with paraformaldehyde solution are damaged after freezing and thawing process to release heme, causing the supernatant to appear red (right side of Figure 1a), and 2.5% (v/v) glutaraldehyde solution is used for treatment Later, the erythrocytes remained intact in shape after freezing and thawing (Figure 1a, left).

Fig. 1b shows the morphology of glutaraldehyde-treated red blood cells observed by SEM, Fig. 1c and Fig. 1d by CLSM. From Figure 1b, Figure 1c and Figure 1d, it can be seen that after using the red blood cell sizing solution of the present invention containing glutaraldehyde for treatment, freezing and thawing, the red blood cell membrane remains intact, and the entire cell shape is elliptical, which can be used for subsequent Single cell detection.

It is further proved that the use of glutaraldehyde solution with a concentration of 2%, 2.5%, 3%, 5% (v/v), especially a concentration of 2.5% (v/v) as a red blood cell sizing solution can achieve complete preservation of red blood cells for a long time. It is speculated that the reason is that the glutaraldehyde solution affects the protein configuration by forming intermolecular cross-links to fix the cell morphology and structure.

Example 2

In this example, the applicable osmic acid labeling concentration for determining the element distribution of a single cell was studied.

1) Take blood: prepare 3 female Balb/c mice, and take the whole blood of the mice in a heparin sodium anticoagulation tube;

2) Add 0.1ml of the above-mentioned whole blood to 0.3ml of PBS and mix well to obtain 0.4ml of cell suspension;

3) Add 1.6ml of glutaraldehyde solution with a concentration of 2.5% (v/v) dropwise to it, while continuing to vortex the cell suspension, and let it stand at room temperature for 20 minutes;

4) Wash the cells twice with PBS by centrifugation (800G, 5min), and discard the supernatant;

5) Suspend all cells in 1ml staining buffer and store in a refrigerator at -80℃;

6) Take out the frozen cells, thaw them naturally at room temperature, centrifuge (800G, 5min), and discard the supernatant;

7) Take about 4×10 ⁶ cells obtained by thawing, suspend the cells in 50μl staining buffer, add 50μl staining buffer containing 1.1μl anti-mouse Ter119-154Sm antibody (3154005B, Fuluda), and mix well , Let stand for 30min at room temperature;

8) Wash the cells twice with staining buffer centrifugation (800G, 5min), discard the supernatant, and disperse it in PBS at a density of ^{about 4×10 6 cells per 100 μl;}

^{9) Add 1ml of 5×10 -6} %, 10 ^-5 %, 3×10 ^-5% , 5×10 ^-5 %, 7×10 ^-5 %, 10% to 100μl of the above cell suspension in PBS. ^-4 %, 5×10 ^-5 % (w/v) osmium acid solution, while continuing to vortex the cell suspension, let it stand at room temperature for 10 minutes;

10) Wash the cells twice with staining buffer by centrifugation (800G, 5min), and discard the supernatant;

11) Suspend the cells in 1ml staining buffer and store in a refrigerator at -80°C;

12) Before using mass spectrometry flow cytometry (Helios) to detect the lead element in a single red blood cell, the cells frozen at -80°C were naturally thawed, and the cells were washed 3 times with ultrapure water by centrifugation (800G, 5min). kind. The internal reference uses EQbeads.

Mass spectrometry flow cytometer detection parameters:

Cell suspension injection flow rate: 30～31μl/min,

Event collection rate <500 events/s,

EQbeads concentration: 10%-15%,

EQbeads monitoring elements: 140/142Ce (cerium), 151/153Eu (europium), 165Ho (holmium), 175/176Lu (lutetium);

Elements to be tested: ¹⁵⁴ Sm (Ter119), ⁷⁸ Se, ⁸⁸ Sr, ¹²⁷ I, ¹⁹² Os, ¹⁹⁰ Os, ²⁰⁸ Pb, ²⁰⁹ Bi.

The test results are shown in Figure 2.

Result: In Figure 2a, the Y-axis is the proportion of single cells labeled with 7 different osmic acid concentrations, and the X-axis is the osmic acid concentration. Among them, it can be seen that at a ^{concentration of 5×10 -6} % osmium acid, the proportion of stained single cells is significantly lower than other concentrations, which proves that this concentration has poor staining effect and is not suitable as a condition for single cell detection.

Figures 2b-2h are scatter plots of flow cytometry. Each point in the figure represents a cell. A darker color represents a high cell density, and a lighter color represents a low cell density. The scatter diagram further confirms that when the concentration of osmic acid is 5×10 ^-6 % (w/v), the cell population is dispersed, which confirms that the staining effect of this concentration is poor; when the concentration of osmic acid solution is 5×10 ^-4 % (w/v) ), the upper end of the cell population shifts to the right, indicating that the staining signal is too strong, affecting the distribution and identification of a single cell population, confirming that the concentration effect is not suitable for single cell detection.

In summary, when the working concentration of osmium acid in the staining solution of the present invention is in the range of 10 ^-5 % to 10 ^-4 % (w/v), it can be used in combination with the erythrocyte-specific metal-labeled antibody as the metal-labeled antibody. Good staining and detection results. Among them, when the concentration is 5×10 ^-5 % (w/v), the distribution of single cells is concentrated, and it can be clearly seen that there are two cell groups. The osmic acid signal of the cell group on the right (shown in a circle) is approximately The 2 times on the left indicates that the cell population on the right is a cell dimer, which distinguishes single cells from dimers and multi-cell clusters, so it is more preferred.

Example 3

The stability of red blood cells treated with red blood cell sizing solution over time.

1) Take 0.1ml of fresh human anticoagulated whole blood, add it to 0.3ml volume of PBS, and mix well to obtain 0.4ml cell suspension;

2) Add 1.6ml of glutaraldehyde solution with a concentration of 2.5% (v/v) dropwise to it, while continuing to vortex the cell suspension, and let it stand at room temperature for 20 minutes;

4) Suspend all the cells in 1ml staining buffer, divide them into 2 parts, store them in a refrigerator at -80°C for later use;

5) Take out a portion of frozen cells when the storage time is 15 days and 150 days respectively, thawed naturally at room temperature, centrifuged (800G, 5min), discarded the supernatant for subsequent labeling;

6) Take about 4×10 ⁶ cells after resuscitation, suspend the cells in 50μl staining buffer, add 50μl staining buffer containing 1.1μl anti-human CD235ab-141Pr antibody (3141001B, Fuluda), mix well, room temperature Let stand for 30 minutes;

7) Wash the cells twice with staining buffer centrifugation (800G, 5min), discard the supernatant, and disperse it in PBS at a density of ^{4×10 6 cells per 100 μl;}

^{8) Add 1 ml of osmium acid solution with a concentration of 5×10 -5} % (w/v) to 100 μl of the above cell suspension in PBS, while continuing to vortex the cell suspension, and let it stand at room temperature for 10 minutes;

9) Wash the cells twice with staining buffer by centrifugation (800G, 5min), and discard the supernatant;

10) Suspend the cells in 1ml staining buffer and store in a refrigerator at -80℃;

11) After 24 hours, the cells frozen at -80°C were naturally thawed, and the cells were washed 3 times by centrifugation (800G, 5min) with ultrapure water, and then loaded. Mass spectrometry flow cytometry (Helios) was used to detect the concentration of single red blood cells. Praseodymium ¹⁴¹ Pr (CD235ab), selenium ⁷⁸ Se, strontium ⁸⁸ Sr, iodine ¹²⁷ I, osmium ¹⁹² Os, osmium ¹⁹⁰ Os, lead ²⁰⁸ Pb, and bismuth 209Bi elements. The internal reference uses EQbeads.

Mass spectrometry flow cytometer detection parameters:

Cell suspension injection flow rate: 30～31μl/min,

Event collection rate <500 events/s,

EQbeads concentration: 10%-15%,

Measured elements: praseodymium ¹⁴¹ Pr (CD235ab), selenium ⁷⁸ Se, strontium ⁸⁸ Sr, iodine ¹²⁷ I, osmium ¹⁹² Os, osmium ¹⁹⁰ Os, lead ²⁰⁸ Pb, bismuth ²⁰⁹ Bi.

The test results are shown in Figure 3 (a) ~ (c).

Results: The two-dimensional flow scatter plot showed that the distribution of individual cell populations stored at -80 degrees Celsius for 15 days and 150 days was similar, with no significant difference. At the same time, the proportions of single cells of the two were also very similar, suggesting a longer storage time. The erythrocyte samples of erythrocytes did not change during the preservation process, which proves that the kit can be used to preserve erythrocyte samples for a long time.

Figure 3(d)～(h) respectively show ^{the individual cell distribution diagrams of the elements 78} Se, ⁸⁸ Sr, ¹²⁷ I, ²⁰⁸ Pb, and ²⁰⁹ Bi. Each point in the figure represents a cell, and the element content in each single cell The value of is represented by the color shown by the color bar on the right of each figure. The closer the color is to the red at the upper end of the color bar, the higher the element content of the point, and the closer the color is to the dark blue at the lower end of the color bar, the element content of this point The lower. The results show that there are a few dark red (very high) and orange (higher) dots in praseodymium, selenium, strontium, iodine, and bismuth, and most of the dots are dark blue (very low), suggesting the distribution pattern of these elements in the sample The elements with high content are concentrated in a small number of red blood cells. For ²⁰⁸ Pb, most of the dots are light blue (low) to light yellow (medium), suggesting that the distribution pattern of lead in the sample is low to medium The content of the element is distributed in a large number of red blood cells, which is different from other elements, and its content is higher than other elements as a whole, and the average amount is between 7 to 25 counts (signal intensity).

Example 4

The staining solution kit of the present invention detects the lead distribution of a single cell in the white blood cells (nucleated cells) of the mouse spleen.

6 female Balb/c mice were divided into lead nitrate group (3 mice) and control group (3 mice). Inject 200μl PBS (control group) or 200μl 5mM lead nitrate solution (exposed group) from the tail vein;

After 4 hours of injection, the mice were dissected and the spleens were taken to prepare a single cell suspension of the spleen, and red blood cells were lysed with red blood cell lysate (erythrocyte lysate, Soleibao) to obtain white blood cell suspension;

The above-mentioned leukocyte suspension was equally divided into two parts, and the kit and method of the present invention (hereinafter referred to as the osmium labeling method) were used for labeling and detection respectively. That is, four samples were divided into control group + osmium labeling method, exposure group + osmium labeling method, control group + iridium labeling method, and exposure group + iridium labeling method, and the following tests were performed:

Control group + osmium labeling method, exposure group + osmium labeling method

1) Take 0.4ml white blood cell suspension, add 1.6ml 2.5% (v/v) glutaraldehyde solution dropwise to it, while continuing to vortex the cell suspension, and let it stand at room temperature for 20 minutes;

2) Wash the cells twice with PBS by centrifugation (800G, 5min), and discard the supernatant;

3) Take 4×10 ⁶ cells, suspend the cells in 50μl staining buffer, add 50μl staining buffer containing 1.1μl anti-mouse CD45-147Sm antibody (3147003B, Fluda), mix well, and let stand at room temperature 30min;

4) Wash the cells twice with staining buffer by centrifugation (800G, 5min), discard the supernatant, and disperse it in PBS at a density of ^{about 4×10 6 cells per 100 μl;}

^{5) Add 1ml of osmium acid solution with a concentration of 5×10 -5} % (w/v) to 100μl of the above cell suspension in PBS dropwise, while continuing to vortex the cell suspension, and let it stand at room temperature for 10 minutes;

6) Wash the cells twice with staining buffer by centrifugation (800G, 5min), discard the supernatant;

7) Use ultrapure water to wash the cells three times by centrifugation (800G, 5min) and load the sample; use mass spectrometry flow cytometry to detect lead. The internal reference uses EQbeads.

Mass spectrometry flow cytometry (Helios) detection parameters

Cell suspension injection flow rate: 30～31μl/min,

Event collection rate <500 events/s,

EQbeads concentration: 10%-15%,

EQbeads monitoring elements: 140/142Ce (cerium), 151/153Eu (europium), 165Ho (holmium), 175/176Lu (lutetium); elements to be tested include: ¹⁵⁴ samarium (Ter119), osmium ¹⁹² Os, osmium ¹⁹⁰ Os, lead ²⁰⁸ Pb.

Control group + iridium labeling method, exposure group + iridium labeling method

1) Take 4×10 ⁶ cells, suspend the cells in 50μl staining buffer, add 50μl staining buffer containing 1.1μl anti-mouse CD45-147Sm antibody (3147003B, Fuluda), mix well, and let stand at room temperature 30min;

2) Wash the cells twice with staining buffer by centrifugation (800G, 5min), discard the supernatant, and disperse it in PBS at a density of ^{about 4×10 6 cells per 100 μl;}

3) In accordance with the commercial kit (201192B, FLUIDIGM) instructions for iridium element labeling;

4) Use mass spectrometry flow cytometry to detect lead element; use Eqbeads as internal reference.

Mass spectrometry flow cytometry (Helios) detection parameters

Cell suspension injection flow rate: 30～31μl/min,

Event collection rate <500 events/s,

EQbeads concentration: 10%-15%,

EQbeads monitoring elements: 140/142Ce (cerium), 151/153Eu (europium), 165Ho (holmium), 175/176Lu (lutetium); the elements to be tested include: ¹⁵⁴ samarium (Ter119), iridium ¹⁹¹ Ir, iridium ¹⁹³ Ir, lead ²⁰⁸ Pb.

The test results are shown in Figure 4, Figure 4(a) shows the proportion of single cells in white blood cells: Among the white blood cells in the control group, the proportion of single cells obtained by the osmium labeling method is higher than that obtained by the iridium labeling method. In the exposed group, Similarly, the proportion of single cells obtained by the osmium labeling method is higher than that of the iridium labeling method.

Figure 4(b) shows the proportion of lead-containing single cells in white blood cells. It can be seen that in the control group not exposed to exogenous lead, the osmium labeling method can more sensitively detect the lead-containing single cells in the mouse leukocytes; in the exposed group, the osmium labeling method and the iridium labeling method can be obtained. The proportion of lead-containing single cells in the white blood cells is similar, suggesting that the osmium labeling method is not only able to label red blood cells, but also an effective method for labeling white blood cells.

The above results indicate that the kit and the detection method of the present invention can also be used for labeling nucleated cells. In addition, the kit and the detection method of the present invention have a comparable labeling effect with commercial iridium element labeling.

The specific embodiments of the present invention further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Industrial applicability

Compared with the traditional total detection technology, the staining solution, the kit and the detection method using it of the present invention solve the detection problem of the average element content in single cells, especially red blood cells, and by using multiple antibodies at the same time, it is expected to achieve simultaneous detection The information of dozens of elements in a single red blood cell provides technical support for scientific research purposes and environmental pollution monitoring. The staining solution, the kit and the detection method using the staining solution, the metal-labeled antibody, and the red blood cell preservation solution in combination of the present invention can realize the determination of various indicators after long-term cryopreservation of red blood cells, and can be widely used for scientific research purposes, Environmental protection, monitoring field, biological sample detection, drug development, etc. of heavy metal pollution.

Claims

A staining solution for labeling cells (such as red blood cells or white blood cells) in mass spectrometry flow cytometry, which contains osmium acid, and at the working concentration of the staining solution, the concentration of the osmium acid is 10 -5 % to 10 -4 %(W/v), preferably 3×10 -5 % to 7×10 -5 % (w/v), more preferably 5×10 -5 % to 7×10 -5 % (w/v).
The kit, comprising the staining solution of claim 1, further comprising: a metal-labeled antibody, preferably a cell-specific metal-labeled antibody.
The kit according to claim 1 or 2, wherein: when the cells are red blood cells, the kit further comprises a red blood cell sizing solution, and the red blood cell sizing solution contains a concentration of 2% to 5% (v/v) , Preferably 2% to 3% (v/v) glutaraldehyde.
The kit according to any one of claims 2 to 3, wherein the cell-specific metal-labeled antibody is a lanthanide-labeled antibody (preferably anti-mouse Ter119-lanthanide-labeled antibody, anti-human CD235ab- One or more of lanthanide-labeled antibody, anti-mouse CD45-lanthanide-labeled antibody, or anti-human CD45-lanthanide-labeled antibody), optionally, a common lanthanide label for detection The lanthanides in antibodies are different from each other.
The kit according to claim 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 (Eu), Gd (Gadolinium), Tb (Tb), Dy (Dysprosium), Ho (Holmium), Er (Erbium), Tm (Thulium), Yb (Ytterbium), Lu (Lutetium) , Scandium (Sc), yttrium (Y), preferably Sm (samarium), Pr (praseodymium).
The kit according to any one of claims 2 to 5, further comprising a staining buffer, the staining buffer containing 0.5% to 1% (w/v) of occluded protein (such as bovine serum white Protein) and a buffer containing no calcium and magnesium ions (preferably a phosphate buffer with a pH range of 7.2 to 7.4, more preferably PBS).
The kit according to claim 2 to 6, further comprising a cell wash solution (preferably a phosphate buffer with a pH range of 7.2 to 7.4, more preferably PBS) does not contain calcium and magnesium ions Preferably, the concentration of the phosphate buffer without the magnesium ion is 0.0067M.
The use of the kit according to any one of claims 2 to 7 in the detection of a single cell, wherein the cells to be detected preferably include red blood cells.
A method for detecting single cell elements, including the following steps:

Treat the cells with a glutaraldehyde solution with a concentration of 2% to 5% (v/v), preferably 2% to 3% (v/v);

The concentration of osmic acid used is 10 -5 ～10 -4% (w/v), preferably 3×10 -5 ～7×10 -5 % (w/v), more preferably 5×10 -5 ～7 ×10 -5 % (w/v) staining solution to label cells (wherein, the concentration of cells is preferably 2×10 6 to 4×10 6 cells/ml);

The cells are labeled with a metal-labeled antibody (preferably a cell-specific metal-labeled antibody).
The method according to claim 9, further comprising one or more of the following steps:

Dissolving the metal-labeled antibody in a staining buffer, the staining buffer being a buffer containing 0.5% to 1% (w/v) blocked protein and free of calcium and magnesium ions;

Washing the cells with the staining buffer;

The cells are washed with a cell wash, which is a phosphate buffer solution without calcium and magnesium ions.