Method for detecting T lymphocyte subtype intracellular factors in rat spleen

Abstract

The invention provides a method for detecting T lymphocyte subtype intracellular factors in the spleen of a rat, and belongs to the technical field of biological detection. The detection method for the T lymphocyte subtype intracellular factors in the rat spleen is constructed by using a fluorescent antibody combination with a specific variety and a specific ratio. Experiments prove that the detection method provided by the invention has excellent detection accuracy and relatively high intra-batch precision and inter-batch precision, can be used for evaluating the influence of drugs on the immune performance of the spleen of the rat in a non-clinical research stage of new drugs, and provides important data for safety and effectiveness evaluation of the drugs.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for detecting intracellular factors of T lymphocyte subtypes in rat spleen. Background Art

[0002] T lymphocytes synthesize many lymphokines and many other cytokines, some of which are related to the treatment of tumors, such as interferon, transfer factor, interleukin, etc. Some of these lymphokines can kill tumor cells, and some can stimulate hematopoietic tissue to counteract the side effects of chemotherapy or radiotherapy. The spleen has 25% of the body's circulating T lymphocytes, directly participates in cellular immunity, and has an important regulatory effect on the distribution of T cell subsets in peripheral blood. The spleen's regulatory effect on T lymphocyte immunity is an important part of tumor immunity. Detection of lymphokines in the spleen can be used to evaluate immune function, diagnosis and differential diagnosis, monitor disease progression, evaluate the effectiveness of drug treatment, formulate individualized treatment plans, and evaluate prognosis.

[0003] Rats are one of the most important scientific research experimental animals. They can be used as model animals for biological products in the non-clinical research stage of new drugs, which helps to clarify the pharmacological and toxicological characteristics. Therefore, it is of great significance to monitor the intracellular factors of T lymphocyte subtypes in the spleen of rats. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting intracellular factors of T lymphocyte subtypes in rat spleen, which is more accurate.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting intracellular factors of T lymphocyte subtypes in rat spleen, comprising the following steps:

[0007] The spleen tissue is sequentially ground, filtered and cell separated to obtain a sample to be tested;

[0008] Perform sample processing, including live and dead staining, surface staining, cell membrane permeabilization and intracellular staining;

[0009] The surface staining comprises taking a combination of antibodies 1 with different fluorescent labels and mixing and incubating them with the sample to be detected;

[0010] Through flow cytometry detection, the detection data is obtained;

[0011] Obtaining T lymphocyte subtype intracellular factors in the spleen of rats according to the detection data;

[0012] The antibody combination 1 includes: CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody.

[0013] Preferably, the fluorescent marker of the CD45 fluorescent antibody is BUV395;

[0014] The fluorescent label of the CD3 fluorescent antibody is FITC;

[0015] The fluorescent label of the CD4 fluorescent antibody is BB700;

[0016] The fluorescent marker of the CD8a fluorescent antibody is BV605;

[0017] Preferably, the volume ratio of the CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody is 0.5-2:1-3:2-3:0.5-2.

[0018] Preferably, the viability staining uses Fixable Viability Stain 780.

[0019] Preferably, the surface staining comprises taking a combination of antibodies 2 with different fluorescent labels and incubating them with the sample to be detected;

[0020] The antibody combination 2 includes: IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody.

[0021] Preferably, the fluorescent label of the IL-2 fluorescent antibody is PE;

[0022] The fluorescent label of the TNF-α fluorescent antibody is PE-Cyanine7;

[0023] The fluorescent label of the IFN-γ fluorescent antibody is AlexaFluor 647.

[0024] Preferably, the volume ratio of the IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody is 0.5-1.5:0.5-1.5:0.5-1.5.

[0025] Preferably, obtaining the detection data includes data acquisition and data analysis;

[0026] The data analysis includes selecting CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + subgroup expression.

[0027] Preferably, the grinding is performed by gentle grinding using the flat end of a syringe piston.

[0028] Preferably, the diameter of the spleen tissue is 0.5 to 2 mm.

[0029] Beneficial effects of the present invention:

[0030] The present invention constructs a method for detecting intracellular factors of T lymphocyte subtypes in rat spleen by using a combination of fluorescent antibodies of specific types and ratios. Experiments have shown that the detection method provided by the present invention has excellent detection accuracy, high intra-batch precision and inter-batch precision, and can be used to evaluate the effect of drugs on the immune performance of rat spleen in the non-clinical research stage of new drugs, providing important data for the safety and effectiveness evaluation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A logical relationship diagram used in the embodiment;

[0032] Figure 2 Figure ① shows the test result of grinding the spleen with the flat end of the syringe;

[0033] Figure 3 Figure ② shows the test result of grinding the spleen with the flat end of the syringe;

[0034] Figure 4 Figure ① is the test result of spleen processed by tissue processing instrument;

[0035] Figure 5 Figure ② shows the test results of spleen processed using tissue processor. DETAILED DESCRIPTION

[0036] Summary of the present invention:

[0037] The flat end of a syringe was used to grind the spleen of rats, and mononuclear cells were extracted from the spleen. The cell concentration was adjusted, and specific stimulatory peptides and PMA were used to stimulate the cells to secrete cytokines. A protein transport inhibitor (BFA) was used to block the intracellular cytokines from being transported to the extracellular space mediated by the Golgi apparatus. After a certain period of stimulation and blocking, T lymphocytes were co-labeled with CD45, CD3, CD4, and CD8 fluorescent antibodies. After incubation, the cells were fixed and holes were punched on the cell membrane. IL-2, IFN-γ, and TNF-α fluorescent antibodies were added to label the intracellular cytokines, and the antibodies were allowed to enter the cells through the artificial holes on the cell membrane and bind to the cytokines before being detected on the machine. After the cells to be tested enter the flow cytometer, they form a single cell bundle wrapped in sheath fluid. The cells are irradiated by the excitation light emitted by the exciter in turn, emitting scattered light and fluorescence of different intensities. These emitted lights are collected by the optical microscope system through the filter and reach the photodetector (photomultiplier tube). The fluorescence signal of the photodetector is converted into an electrical signal, which is converted into a digital signal by a digital converter. The lymphocyte population is selected, and the lymphocytes containing cytokines such as IL-2, IFN-γ, and TNF-α are collected through fluorescent antibody labeling, and CD45 is calculated. + CD3 + CD4 + and CD45 + CD3 + CD8 + The expression of IFN-γ, IL-2, and TNF-α on cells.

[0038] The sources of the reagents used in the embodiments of the present invention are shown in Table 1 below:

[0039] Table 1 Example reagent information

[0040]

[0041]

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example

[0044] (1) Remove the spleen and place it on a culture dish. Cut it into pieces of about 1 mm. Add 1 mL of 1×PBS to the culture dish. Use the flat end of the disposable syringe piston to gently grind the tissue in a clockwise direction until only connective tissue remains. Discard the connective tissue, place the cell filter on a fixed centrifuge tube, and pour the cell suspension in the culture dish into the cell filter. Add 500 μL of 1×PBS to rinse the culture dish, filter again, and collect the filtrate. Centrifuge at 400×g, 4°C for 5 minutes, and discard the supernatant.

[0045] (2) Add the cells to 2 mL of saline (tube A) and mix well.

[0046] (3) Slowly add the cells in tube A along the wall of the tube into the tube B containing 3 mL of LSM so that the liquid is spread on the LSM. Centrifuge at 400 × g, 15-25°C for 30 min.

[0047] (4) After centrifugation, a middle white cell layer is visible. Discard the upper liquid layer and transfer the white cell layer to a centrifuge tube (tube C) containing 3 mL of 1× PBS. Mix well and centrifuge at 250×g, 15-25°C for 10 min.

[0048] (5) After centrifugation, discard the supernatant and add 1000 μL of RPMI1640 complete medium to the remaining cells at the bottom of the tube. Mix gently to prepare a cell suspension.

[0049] (6) Take out 50 μL of the cell suspension, dilute it 10 times, and use a fully automatic five-differentiation cell analyzer to detect the white blood cell concentration.

[0050] (7) According to the counting results, the cell suspension was diluted with RPMI1640 complete medium to make the concentration of the diluted cell suspension of each animal 2-2.5×10 6 pcs / mL, for future use.

[0051] Reagent preparation

[0052] (1) Leukocyte Activation Cocktail: Dissolve the leukocyte activation cocktail and store at 2-8°C until ready for use.

[0053] (2) Specific stimulatory peptide: add 15 μL of DMSO and 285 μL of RPMI1640 liquid culture medium to dissolve.

[0054] (3) Protein Transport Inhibitor (Containing Brefeldin A): Store at 2-8°C until used.

[0055] (4) Fixable Viability Stain 780: dilute 1000-fold with 1× PBS and store at 2-8°C until use.

[0056] Cell stimulation and culture

[0057] (1) A negative control group, a positive control group, and a sample peptide stimulation sample group were set up: the negative control group only added Protein Transport Inhibitor (Containing Brefeldin A), the positive control group added Leukocyte Activation Cocktail, and the sample peptide stimulation sample group added specific stimulatory peptide and Protein Transport Inhibitor (Containing Brefeldin A).

[0058] (2) Take each diluted cell suspension sample (concentration of about 2 to 2.5×10 6 / mL) 980 μL was added to the corresponding sample peptide stimulation well plate and negative control well plate. In the positive control group, 1000 μL of 3 cell suspensions were randomly selected and added to the positive control well plate.

[0059] (3) Add 20 μL of specific stimulator peptide (final concentration 2.0 μg / ml / well) to the sample peptide stimulation wells and 2 μL of Leukocyte Activation Cocktail to the positive control wells, and mix gently.

[0060] (4) After adding the sample, place the plate in a carbon dioxide incubator at 37°C, 5% CO2, and saturated humidity for 2 hours for stimulation culture.

[0061] (5) Blocking: After 2 hours of stimulation, remove the sample peptide stimulation plate and the negative control plate, add 1 μL of Protein Transport Inhibitor (containing Brefeldin A) to each well, and continue culturing for 4 hours in a carbon dioxide incubator at 37°C and 5% CO2 with saturated humidity.

[0062] Cell collection

[0063] After the culture is completed, take out the culture well plate, gently blow and mix the cells in the well plate, collect the cells in the well plate into the flow tube, add 1000μL 1×PBS, 400×g, 15-25℃, centrifuge for 5min, discard the supernatant, and leave the cells at the bottom of the tube, add 1000μL 1×PBS again, wash, 400×g, 15-25℃, centrifuge for 5min, discard the supernatant, and leave about 100μL of cells at the bottom of the tube, and mix to form a cell suspension.

[0064] Sample processing and testing

[0065] Dead and alive staining

[0066] (1) Pipette 100 μL of cell suspension into each flow cytometry tube.

[0067] (2) Place 1000 μL of the diluted Fixable Viability Stain 780 in each flow cytometry tube, mix well, and incubate at room temperature in the dark for 10 min.

[0068] (3) Add 1000 μL 1× PBS to each tube, wash and centrifuge at 20°C, 400×g, 5 min, and discard the supernatant.

[0069] FcR blockade

[0070] Take 50 μL of Purified Mouse Anti-Rat CD32 dilution solution into each flow tube and incubate in a dark place at 2-8°C refrigerator for 5 minutes without washing.

[0071] Surface staining

[0072] (1) Staining: Pipette 1.0 μL BUV395 Mouse Anti-Rat CD45, 2.0 μL FITCAnti-Rat CD3, 2.5 μL BBB700 Mouse Anti-Rat CD4, and 1.0 μL BV605 Mouse Anti-Rat CD8 fluorescent antibodies into each flow cytometry tube, mix well, and incubate at 15-25°C in the dark for 20 min.

[0073] (2) Centrifugation after washing: Add 1000 μL 1× PBS for washing. Centrifuge at 20°C, 400×g, for 5 min. Discard the supernatant.

[0074] intracellular staining

[0075] (1) Reagent preparation: Use Diluent Buffer to prepare Fix / Perm Buffer (4×) into Fix / Perm Buffer (1×). Use pure water to prepare Perm / Wash Buffer (5×) into Perm / Wash Buffer (1×). Store at 2-8°C in dark for future use.

[0076] (2) Fixation and permeabilization: Add 1000 μL of TF Fix / Perm Buffer (1×) to each flow cytometry tube, vortex to mix, and incubate at 2-8°C in the dark for 45 min.

[0077] (3) Centrifugation after washing: Add 1000 μL of TF Perm / Wash Buffer (1×) for washing. Centrifuge under the following conditions: temperature 4°C, speed 400×g, time 5 min, and discard the supernatant.

[0078] (4) Centrifugation after washing: Add 2000 μL of TF Perm / Wash Buffer (1×) for washing. Centrifuge under the following conditions: temperature 4°C, speed 400×g, time 5 min, and discard the supernatant.

[0079] (5) Intracellular staining: Add 100 μL TF 1× Perm / Wash Buffer to each flow cytometry tube, and pipette 2.5 μL Anti-IL-2 Antibody (PE), Mouse Monoclonal, 2.5 μL Alexa Fluor 647 Mouse Anti-Rat IFN-γ, and 2.5 μL TNF alpha Monoclonal Antibody, PE-Cyanine 7 fluorescent antibody into each flow cytometry tube, shake to mix, and incubate at 2-8°C in the dark for 45 min.

[0080] (6) Centrifugation after washing: Add 2000 μL of TF Perm / Wash Buffer (1×) for washing. Centrifuge at 4°C, 400×g, for 5 min. Discard the supernatant.

[0081] (7) Sample testing: Take out the centrifuged sample, discard the supernatant, add 500 μL 1× PBS buffer to resuspend, shake to mix, and store in the dark for testing.

[0082] Data processing and analysis

[0083] The samples were tested using the CytoFLEX LX flow cytometer, and the test data were analyzed using the instrument's built-in CytExpert software. The target cell population was determined using a comprehensive method of histograms, scatter plots, and contour plots. Analysis parameters such as fluorescence compensation and gate position were adjusted as needed to confirm the distribution of each cell population and obtain the required data. The % Parent value was obtained as the sample result.

[0084] The instrument parameters are shown in Tables 2 and 3 below:

[0085] Table 2 Instrument voltage, threshold

[0086] Channel Name Voltage Threshold FSC 266 50000 SSC 174 / B525-FITC 189 / B690-PC5.5 588 / Y585-PE 218 / Y763-PC7 93 / R660-APC 306 / R763-APCA750 360 / V610 98 / NUV450 3 /

[0087] Table 3 Instrument compensation

[0088]

[0089]

[0090] Data analysis logic diagram

[0091] The samples were tested using the CytoFLEX LX flow cytometer, and the test data were analyzed using the instrument's built-in CytExpert software to determine the target cell population, adjust the fluorescence compensation, gate position and other analysis parameters, confirm the distribution of each cell population, and obtain the required data. The logical relationship is as follows: Figure 1 shown.

[0092] Comparative Example

[0093] The only difference from the embodiment is that the step of grinding the spleen with the flat end of the syringe is replaced by using a tissue processor to process the spleen. The same test is performed, and the results are as follows Figures 2 to 5 shown.

[0094] The comparison results showed that the survival rate of the spleen was 99% when the flat end of the syringe was used to grind the spleen, and the data results met the requirements; the survival rate was reduced when the spleen was processed by the tissue processor, and CD3 + 、CD4 + 、CD8 + Unknown group, CD4 + / CD8 + The proportions are inverted.

[0095] Experimental example

[0096] Methodological verification of the technical solution provided in the embodiment:

[0097] Intra-run precision (WP): 4 samples were tested in the same analytical batch and the test was repeated 3 times. The %CV of each test value of each sample was calculated, and the %CV was between 0.24 and 14.21% (satisfying CV ≤ 25%). The intra-run precision met the requirements. The specific results are shown in Table 4.

[0098] Between-run Precision (BP): The same three samples were analyzed in two independent analysis batches within one day, and each sample in each batch was tested three times. The %CV of each test value of the same sample in the two analysis batches was calculated, and the %CV was between 0.03 and 16.03% (satisfying CV ≤ 25%). The between-run precision met the requirements. The specific results are shown in Table 5.

[0099] Table 4 Intra-batch precision of flow cytometry for detecting intracellular factors of T lymphocyte subtypes in rat spleen

[0100]

[0101]

[0102]

[0103]

[0104] Table 5 Inter-batch precision of flow cytometry for detecting intracellular factors of T lymphocyte subtypes in rat spleen

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] The verification results showed that the intra-batch precision CV range of the scheme provided by the present invention was 0.24-14.21%; the inter-batch precision CV range was 0.03-16.03%, indicating that the method has accurate detection results and can be used for the detection of intracellular factors of T lymphocyte subtypes in rat spleen.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting intracellular factors of T lymphocyte subtypes in rat spleen, characterized in that: The following steps are involved: The spleen tissue is sequentially ground, filtered and cell separated to obtain a sample to be tested; Perform sample processing, including live and dead staining, surface staining, cell membrane permeabilization and intracellular staining; The surface staining comprises taking a combination of antibodies 1 with different fluorescent labels and mixing and incubating them with the sample to be detected; Through flow cytometry detection, the detection data is obtained; Obtaining T lymphocyte subtype intracellular factors in the spleen of rats according to the detection data; The antibody combination 1 includes: CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody.

2. The detection method according to claim 1, characterized in that: The fluorescent marker of the CD45 fluorescent antibody is BUV395; The fluorescent label of the CD3 fluorescent antibody is FITC; The fluorescent label of the CD4 fluorescent antibody is BB700; The fluorescent marker of the CD8a fluorescent antibody is BV605.

3. The detection method according to claim 2, characterized in that: The volume ratio of the CD45 fluorescent antibody, CD3 fluorescent antibody, CD4 fluorescent antibody and CD8a fluorescent antibody is 0.5-2:1-3:2-3:0.5-2.

4. The detection method according to claim 1, characterized in that: The described dead-live staining adopts FixableViability Stain 780.

5. The detection method according to claim 1, characterized in that: The surface staining comprises taking a combination of antibodies 2 with different fluorescent labels and mixing and incubating them with the sample to be detected; The antibody combination 2 includes: IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody.

6. The detection method according to claim 5, characterized in that: The fluorescent label of the IL-2 fluorescent antibody is PE; The fluorescent label of the TNF-α fluorescent antibody is PE-Cyanine7; The fluorescent label of the IFN-γ fluorescent antibody is AlexaFluor 647.

7. The detection method according to claim 5, characterized in that: The volume ratio of the IL-2 fluorescent antibody, TNF-α fluorescent antibody and IFN-γ fluorescent antibody is 0.5-1.5:0.5-1.5:0.5-1.

5.

8. The detection method according to claim 3, characterized in that: The obtaining of detection data includes data acquisition and data analysis; The data analysis includes selecting CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + subgroup expression.

9. The detection method according to claim 1, characterized in that: The grinding is gentle grinding using the flat end of a syringe plunger.

10. The detection method according to claim 1, characterized in that: The diameter of the spleen tissue is 0.5-2 mm.

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