A sample processing reagent and its application in flow cytometry analysis

Abstract

This invention relates to a sample processing reagent and its application in flow cytometry analysis, belonging to the field of flow cytometry analysis technology. The invention provides a sample processing reagent comprising a hemolysin and a cell-permeable agent. The hemolysin comprises tris(hydroxymethyl)aminomethane (Tris), sodium chloride, calcium chloride, disodium ethylenediaminetetraacetate, paraformaldehyde, and a solvent. The cell-permeable agent comprises sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X-100, paraformaldehyde, and a solvent. The hemolysin is mild and highly effective, providing complete hemolysis and clear cell clustering. The cell-permeable agent permeates the cell membrane under isotonic and neutral conditions, minimizing damage to cell surface antigens and effectively detecting both cytoplasmic and nuclear antigens.

Description

Technical Field

[0001] This invention relates to a sample processing reagent and its application in flow cytometry analysis, belonging to the field of flow cytometry analysis technology. Background Technology

[0002] Flow cytometry works by using monoclonal antibodies to perform rapid, multi-parameter quantitative analysis of single cells or other biological particles at the cellular and molecular level. It can analyze tens of thousands of cells at high speed and simultaneously measure multiple parameters from a single cell, offering advantages such as speed, high precision, and good accuracy. Currently, flow cytometry is widely used in oncology, immunology, pharmacology, hematology, genetics, cell biology, and other fields.

[0003] Before performing flow cytometry analysis on samples, pretreatment with hemolysin and permeabilizing agents is typically required. Hemolysin functions to both lyse and fix red blood cells, ensuring that the erythrocytes are fully lysed while preserving the antigenicity of cell structure and proteins. Permeabilizing agents bind to lipids and some proteins on the cell and nuclear membranes, creating pores sufficient for immunofluorescence staining to pass through. This allows the immunofluorescence staining agent to enter the cells and nucleus and specifically bind to antigens within the cytoplasm and nucleus. The combined action of hemolysin and permeabilizing agents enables the detection of antigens in the cytoplasm and nucleus using flow cytometry.

[0004] However, existing hemolytic agents have problems such as incomplete hemolysis or poor cell clustering, and existing membrane-breaking agents have problems such as weak membrane-breaking effect or excessive damage to cell surface antigens. These problems make it difficult to accurately select the target cell population and simultaneously and completely detect cell surface and intracellular antigens when using flow cytometry to analyze the sample. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a sample processing reagent comprising a hemolysin, a membrane-breaking agent, and a washing solution; the hemolysin comprises tris(hydroxymethyl)aminomethane (Tris), sodium chloride, calcium chloride, disodium ethylenediaminetetraacetate, paraformaldehyde, and a solvent; the membrane-breaking agent comprises sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X-100, paraformaldehyde, and a solvent.

[0006] In one embodiment of the present invention, the washing solution comprises sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20, and a solvent.

[0007] In one embodiment of the present invention, the concentration of tris(hydroxymethyl)aminomethane in the hemolytic agent is 1-2 g / L, the concentration of sodium chloride is 0.5-1 g / L, the concentration of calcium chloride is 0.1-0.5 g / L, the concentration of disodium ethylenediaminetetraacetate is 0.5-1 g / L, and the concentration of paraformaldehyde is 10-15 g / L.

[0008] In one embodiment of the present invention, the concentration of tris(hydroxymethyl)aminomethane in the hemolytic agent is 1.58 g / L, the concentration of sodium chloride is 0.58 g / L, the concentration of calcium chloride is 0.33 g / L, the concentration of disodium ethylenediaminetetraacetate is 0.67 g / L, and the concentration of paraformaldehyde is 12.5 g / L.

[0009] In one embodiment of the present invention, the concentration of sodium chloride in the film-breaking agent is 8-12 g / L, the concentration of disodium hydrogen phosphate is 1-1.5 g / L, the concentration of sodium dihydrogen phosphate is 0.1-0.3 g / L, the concentration of nonylphenol polyoxyethylene ether is 1-2 g / L, the concentration of Triton X-100 is 0.3-0.8 g / L, and the concentration of paraformaldehyde is 20-30 g / L.

[0010] In one embodiment of the present invention, the film-breaking agent contains sodium chloride at a concentration of 9 g / L, disodium hydrogen phosphate at a concentration of 1.15 g / L, sodium dihydrogen phosphate at a concentration of 0.23 g / L, nonylphenol polyoxyethylene ether at a concentration of 1.59 g / L, Triton X-100 at a concentration of 0.53 g / L, and paraformaldehyde at a concentration of 25 g / L.

[0011] In one embodiment of the present invention, the concentration of sodium chloride in the washing solution is 5-10 g / L, the concentration of potassium chloride is 0.1-0.3 g / L, the concentration of disodium hydrogen phosphate is 1-2 g / L, the concentration of potassium dihydrogen phosphate is 0.1-0.3 g / L, and the concentration of Tween 20 is 0.3-0.8 g / L.

[0012] In one embodiment of the present invention, the washing solution contains sodium chloride at a concentration of 8 g / L, potassium chloride at a concentration of 0.2 g / L, disodium hydrogen phosphate at a concentration of 1.42 g / L, potassium dihydrogen phosphate at a concentration of 0.24 g / L, and Tween 20 at a concentration of 0.55 g / L.

[0013] In one embodiment of the present invention, the solvent is water.

[0014] The present invention also provides a hemolytic agent comprising tris(hydroxymethyl)aminomethane (Tris), sodium chloride, calcium chloride, disodium ethylenediaminetetraacetate, paraformaldehyde, and a solvent.

[0015] In one embodiment of the present invention, the concentration of tris(hydroxymethyl)aminomethane in the hemolytic agent is 1.58 g / L, the concentration of sodium chloride is 0.58 g / L, the concentration of calcium chloride is 0.33 g / L, the concentration of disodium ethylenediaminetetraacetate is 0.67 g / L, and the concentration of paraformaldehyde is 12.5 g / L.

[0016] In one embodiment of the present invention, the solvent is water.

[0017] The present invention also provides a film-breaking agent, the components of which include sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X-100, paraformaldehyde, and a solvent.

[0018] In one embodiment of the present invention, the film-breaking agent contains sodium chloride at a concentration of 9 g / L, disodium hydrogen phosphate at a concentration of 1.15 g / L, sodium dihydrogen phosphate at a concentration of 0.23 g / L, nonylphenol polyoxyethylene ether at a concentration of 1.59 g / L, Triton X-100 at a concentration of 0.53 g / L, and paraformaldehyde at a concentration of 25 g / L.

[0019] In one embodiment of the present invention, the solvent is water.

[0020] The present invention also provides a washing solution, the components of which include sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and a solvent.

[0021] In one embodiment of the present invention, the washing solution contains sodium chloride at a concentration of 8 g / L, potassium chloride at a concentration of 0.2 g / L, disodium hydrogen phosphate at a concentration of 1.42 g / L, potassium dihydrogen phosphate at a concentration of 0.24 g / L, and Tween 20 at a concentration of 0.55 g / L.

[0022] In one embodiment of the present invention, the solvent is water.

[0023] The present invention also provides a flow cytometry analysis method, wherein the flow cytometry analysis method uses the above-mentioned sample processing reagent to process the sample to be tested.

[0024] In one embodiment of the present invention, the flow cytometry analysis method includes the following steps:

[0025] Hemolysis procedure: The sample to be tested is mixed with a hemolytic agent and incubated to obtain an incubation solution; the incubation solution is centrifuged, the precipitate is washed with washing solution to obtain the hemolysis product;

[0026] Membrane disruption steps: Mix the hemolysate with the membrane disruption agent and incubate to obtain an incubation solution; centrifuge the incubation solution, take the precipitate and wash it with washing solution to obtain the membrane disruption product;

[0027] Staining steps: Mix the membrane-perforated product with the immunofluorescence staining agent and incubate to obtain the incubation solution; centrifuge the incubation solution, take the precipitate and wash it with the washing solution to obtain the stained product;

[0028] Analysis steps: The stained products were analyzed using flow cytometry.

[0029] The present invention also provides the application of the above-mentioned sample processing reagents, hemolysins, membrane-breaking agents, washing solutions, or flow cytometry methods in flow cytometry analysis.

[0030] The technical solution of this invention has the following advantages:

[0031] This invention provides a sample processing reagent comprising a hemolysin, a cell-permeable agent, and a washing solution. The hemolysin comprises tris(hydroxymethyl)aminomethane (Tris), sodium chloride, calcium chloride, disodium ethylenediaminetetraacetate, paraformaldehyde, and a solvent. The cell-permeable agent comprises sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X-100, paraformaldehyde, and a solvent. The hemolysin is mild and highly effective, providing complete hemolysis and clear cell clustering. The cell-permeable agent permeates cells under isotonic and neutral conditions, minimizing damage to cell surface antigens and effectively detecting both cytoplasmic and nuclear antigens. Attached Figure Description

[0032] Figure 1 : Analyze the FSvsSS plot of lymphocyte phylum and non-fragmentation phylum.

[0033] Figure 2 Analysis of the non-fragmented gate CD4 vs SS diagram.

[0034] Figure 3 : Analyze the CD4 vs SS diagram of lymphocyte phylum.

[0035] Figure 4 : Analysis of CD4 vs Foxp3 diagrams of lymphocyte phyla.

[0036] Figure 5 : Analysis of CD4 + A single-parameter graph of Foxp3 in lymphocyte phyla.

[0037] Figure 6 Analysis of CD4 vs CD25 in lymphocyte phylum.

[0038] Figure 7 : Analysis of CD4 + Diagram of CD25 vs Foxp3 in lymphocyte phylum. Detailed Implementation

[0039] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0040] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0041] The flow cytometers used in the following examples were purchased from BD Biosciences, and the following reagents were purchased from Sinopharm Group: Tris, sodium chloride, calcium chloride, disodium ethylenediaminetetraacetate, paraformaldehyde, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X-100 and purified water. The immunofluorescence staining agents were purchased from Thermo Fisher Scientific.

[0042] Example 1: A sample processing reagent

[0043] This embodiment provides a sample processing reagent, which consists of a hemolysin (solution A), a membrane-breaking agent (solution B), and a washing solution (solution C). The hemolysin comprises tris(hydroxymethyl)aminomethane (Tris), sodium chloride, calcium chloride, disodium ethylenediaminetetraacetate (EDTA), paraformaldehyde, and purified water. Specifically, the concentrations of Tris(hydroxymethyl)aminomethane, sodium chloride, calcium chloride, disodium ethylenediaminetetraacetate, and paraformaldehyde in the hemolysin are 1.58 g / L, 0.58 g / L, 0.33 g / L, 0.67 g / L, and 12.5 g / L. The membrane-breaking agent comprises sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X-100, and poly(hydroxymethyl)acetate. The sample preparation solution consists of polyoxymethylene and purified water. The film-breaking agent contains sodium chloride at a concentration of 9 g / L, disodium hydrogen phosphate at a concentration of 1.15 g / L, sodium dihydrogen phosphate at a concentration of 0.23 g / L, nonylphenol polyoxyethylene ether at a concentration of 1.59 g / L, Triton X-100 at a concentration of 0.53 g / L, and paraoxymethylene at a concentration of 25 g / L. The washing solution consists of sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20, and purified water. The washing solution contains sodium chloride at a concentration of 8 g / L, potassium chloride at a concentration of 0.2 g / L, disodium hydrogen phosphate at a concentration of 1.42 g / L, potassium dihydrogen phosphate at a concentration of 0.24 g / L, and Tween 20 at a concentration of 0.55 g / L. The preparation method of the sample processing reagents is shown in Table 1.

[0044] Table 1. Preparation method of sample processing reagents

[0045]

[0046] Example 2: A flow cytometry method

[0047] This embodiment provides a flow cytometry analysis method. The flow cytometry analysis method uses the sample processing reagent of Example 1 to process the sample to be tested. The washing buffer is diluted with purified water at a volume ratio of 1:9 (wash buffer: purified water = 1:9) to prepare a working solution before use. The method includes the following steps:

[0048] Hemolysis procedure: Mix 100 μL of the test sample with 2 mL of hemolysing agent and incubate at room temperature (25℃) in the dark for 10 min to obtain the incubation solution; centrifuge the incubation solution at room temperature (25℃) and 300 x g for 5 min, then resuspend the precipitate in the working solution obtained by diluting it with 2 mL of washing buffer to obtain the resuspended solution; centrifuge the resuspended solution at room temperature (25℃) and 300 x g for 5 min, then collect the precipitate to obtain the hemolysis product;

[0049] Membrane disruption steps: Mix the hemolysate with 1 mL of membrane disruption agent and incubate at room temperature (25℃) in the dark for 60 min to obtain the incubation solution; centrifuge the incubation solution at room temperature (25℃) and 300 x g for 5 min, then resuspend the precipitate in the working solution obtained by diluting it with 2 mL of washing buffer to obtain the resuspension; centrifuge the resuspension at room temperature (25℃) and 300 x g for 5 min, then collect the precipitate to obtain the membrane disruption product;

[0050] Staining procedure: Mix the membrane-breaking product with 15 μL of immunofluorescence staining agent and incubate at room temperature (25℃) in the dark for 60 min to obtain the incubation solution; centrifuge the incubation solution at room temperature (25℃) and 300 x g for 5 min, then resuspend the precipitate in the working solution obtained by diluting it with 2 mL of washing buffer to obtain the resuspended solution; centrifuge the resuspended solution at room temperature (25℃) and 300 x g for 5 min, then collect the precipitate to obtain the staining product;

[0051] Analytical procedure: The staining product was diluted with 0.5 mL of washing buffer to obtain the working solution, and then analyzed using flow cytometry to obtain the analytical results.

[0052] Experiment Example 1: Verification of the effectiveness of sample treatment reagents

[0053] Anticoagulated whole blood (from healthy individuals) was used as the test sample, and eBioscience was used to analyze it. TMFoxp3 / Transcription Factor Staining Buffer Set (purchased from Thermofisher-Invitrogen, model 00-5523-00) and Transcription Factor Buffer Set (purchased from BD, model 562574) were used as controls A and B, respectively. Flow cytometry analysis of CD4, CD25, and Foxp3 in the test samples was performed using the method described in Example 2. The results are shown in [Figure 1]. Figures 1-7 .

[0054] Depend on Figures 1-7 It can be seen that the sample processing reagent used in Example 1 resulted in complete hemolysis, clear cell clustering, intact preservation of cell surface antigens, good membrane perforation effect, high detection rate of intracellular antigens, and high fluorescence intensity. In contrast, the sample processing reagent used in the control group resulted in poor hemolysis effect, large changes in cell morphology, unclear cell clustering, partial or complete loss of cell surface antigens, unsatisfactory membrane perforation effect, low detection rate of intracellular antigens, and low fluorescence intensity.

[0055] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A sample processing reagent, characterized by, The sample processing reagent comprises a hemolytic agent, a membrane breaking agent and a washing solution; the hemolytic agent comprises Tris, sodium chloride, calcium chloride, disodium EDTA, paraformaldehyde and a solvent; the membrane breaking agent comprises sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X-100, paraformaldehyde and a solvent; the washing solution comprises sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, Tween 20 and a solvent; In the hemolytic agent, the concentration of Tris is 1-2 g / L, the concentration of sodium chloride is 0.5-1 g / L, the concentration of calcium chloride is 0.1-0.5 g / L, the concentration of disodium EDTA is 0.5-1 g / L, and the concentration of paraformaldehyde is 10-15 g / L; in the membrane breaking agent, the concentration of sodium chloride is 8-12 g / L, the concentration of disodium hydrogen phosphate is 1-1.5 g / L, the concentration of sodium dihydrogen phosphate is 0.1-0.3 g / L, the concentration of nonylphenol polyoxyethylene ether is 1-2 g / L, the concentration of Triton X-100 is 0.3-0.8 g / L, and the concentration of paraformaldehyde is 20-30 g / L; in the washing solution, the concentration of sodium chloride is 5-10 g / L, the concentration of potassium chloride is 0.1-0.3 g / L, the concentration of disodium hydrogen phosphate is 1-2 g / L, the concentration of potassium dihydrogen phosphate is 0.1-0.3 g / L, and the concentration of Tween 20 is 0.3-0.8 g / L.

2. A flow cytometric analysis method characterized by, The flow cytometry analysis method uses the sample processing reagent of claim 1 to process the sample to be tested.

3. Use of the sample processing reagent of claim 1 or the flow cytometry analysis method of claim 2 in flow cytometry analysis.

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