CN119804878A - A method for detecting intracellular factors of T lymphocyte subtypes in peripheral blood of rhesus monkeys - Google Patents

Abstract

The present invention provides a method for detecting intracellular factors of T lymphocyte subtypes in the peripheral blood of rhesus monkeys, and belongs to the field of biological detection technology. The present invention constructs a method for detecting intracellular factors of T lymphocyte subtypes in the peripheral blood of rhesus monkeys by using a combination of fluorescent antibodies of specific types and ratios. Experiments have proved 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 rhesus monkeys in the non-clinical research stage of new drugs, providing important data for the safety and effectiveness evaluation of drugs.

Description

Method for detecting T lymphocyte subtype intracellular factors in peripheral blood of rhesus monkey

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for detecting T lymphocyte subtype intracellular factors in peripheral blood of rhesus monkeys.

Background

T lymphocytes synthesize many lymphokines, and many other cytokines, some of which are associated with the treatment of tumors, such as interferons, metastasis factors, interleukins, etc., some of which kill tumor cells, some of which stimulate hematopoietic tissues, and others of which are resistant to the side effects of chemotherapy or radiation therapy. The spleen has 25% of the total circulating T lymphocytes, directly participates in cellular immunity, and has important regulation effect on the distribution of T cell subsets in peripheral blood. The regulatory effect of spleen on T lymphocyte immunity is an important link of tumor immunity. The detection of lymphokines in the spleen can be used to assess immune function, diagnose and differential diagnosis, monitor disease progression, evaluate drug treatment efficacy, personalized treatment regimen formulation, prognostic assessment, and the like.

Rhesus monkey is one of the very important scientific experimental animals, and can be used as a model animal of biological products in the non-clinical research stage of new drugs, so that the elucidation of pharmacological and toxicological characteristics is facilitated, and therefore, the monitoring of T lymphocyte subtype intracellular factors in the peripheral blood of the rhesus monkey is of great importance.

Disclosure of Invention

The invention aims to provide a method for detecting T lymphocyte subtype intracellular factors in peripheral blood of rhesus monkeys, which has excellent detection accuracy.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for detecting a rhesus T lymphocyte subtype intracellular factor, which comprises the following steps:

sample processing including dead and alive staining, surface staining, cell rupture and intracellular staining;

the surface staining comprises the steps of taking antibody combinations 1 with different fluorescent labels, and mixing and incubating with a sample to be detected;

detecting through flow cytometry, and obtaining detection data results of the macaque T lymphocyte subtype intracellular factors;

The antibody combination 1 comprises a CD45 fluorescent antibody, a CD3 fluorescent antibody, a CD4 fluorescent antibody and a CD8a fluorescent antibody.

Preferably, the fluorescent label of the CD45 fluorescent antibody is BV510;

the fluorescent label of the CD3 fluorescent antibody is APC;

the fluorescent label of the CD4 fluorescent antibody is BUV395;

the fluorescent label of the CD8a fluorescent antibody is AlexaFluor 700.

Preferably, the volume ratio of the CD45 fluorescent antibody, the CD3 fluorescent antibody, the CD4 fluorescent antibody and the CD8a fluorescent antibody is 0.5-2:8-12:0.5-2:0.5-2.

Preferably, fixableViability Stain 780,780 is used for the dead and alive staining.

Preferably, the cell rupture membrane is TF Fix/PermBuffer.

Preferably, the intracellular staining comprises taking antibody combinations 2 with different fluorescent labels, and mixing and incubating with a sample to be detected;

the antibody combination 2 comprises IL-2 fluorescent antibody, TNF-alpha fluorescent antibody and IFN-gamma fluorescent antibody.

Preferably, the fluorescent label of the IL-2 fluorescent antibody is BV421;

The fluorescent label of the TNF-alpha fluorescent antibody is PE;

the fluorescent label of the IFN-gamma fluorescent antibody is FITC.

Preferably, the volume ratio of the IL-2 fluorescent antibody, the TNF-alpha fluorescent antibody and the IFN-gamma fluorescent antibody is 0.5-1.5:3-5:0.5-1.5.

Preferably, the obtained detection data comprises data acquisition and data analysis;

The data analysis includes selection CD45⁺CD3⁺CD4⁺、CD45⁺CD3⁺CD8⁺、CD45⁺CD3⁺CD4⁺IFN-γ⁺、CD45⁺CD3⁺CD4⁺IL-2⁺、CD45⁺CD3⁺CD4⁺TNF-α⁺、CD45⁺CD3⁺CD8⁺IFN-γ⁺、CD45⁺CD3⁺CD8⁺IL-2⁺、CD45⁺CD3⁺CD8⁺TNF-α⁺ of subpopulation expression profiles.

Preferably, the sample is from rhesus peripheral blood.

The invention has the beneficial effects that:

The invention constructs a detection method of T lymphocyte subtype intracellular factors in peripheral blood of rhesus monkeys by using fluorescent antibody combinations with specific types and proportions. Experiments prove that the detection method provided by the invention has excellent detection accuracy, the in-batch precision and the inter-batch precision are high, the in-batch precision of the positive medicament stimulation experiment set also meets the requirements, and the method can be used for evaluating the influence of medicaments on the immune performance of peripheral blood of rhesus monkeys in a non-clinical research stage of new medicaments, and provides important data for evaluating the safety and effectiveness of the medicaments.

Drawings

FIG. 1 is a diagram of the logical relationship employed by an embodiment;

FIG. 2 is a graph showing the results of negative detection in stimulus-blocking cultures;

FIG. 3 is a graph showing the results of positive stimulation-blocking culture tests.

Detailed Description

Summary of the solution:

Collecting peripheral blood of rhesus monkey, extracting mononuclear cells, regulating cell concentration, stimulating cell secretion of cytokines by using specific stimulator polypeptide and phorbol myristate 13-acetate (PMA)/Ionomycin (Ionomycin), blocking intracellular cytokines by using protein transport inhibitor Brufilidin A (BFA) to be transported out of the cell by mediation of a golgi body, jointly marking T lymphocytes by using CD45, CD3, CD4 and CD8 fluorescent antibodies after blocking for a certain time, fixing the cells after incubation, punching on the cell membrane, adding IL-2, IFN-gamma and TNF-alpha fluorescent antibodies to mark the intracellular cytokines, enabling the antibodies to enter the cell through artificial small holes on the cell membrane to be combined with the cytokines, and then performing on-line detection. After entering the flow cytometer, the cells to be detected form single cell beams wrapped by sheath fluid, the cells are irradiated by excitation light emitted by an exciter in sequence, scattered light and fluorescence with different intensities are emitted, the emitted light is collected by a light filter system through a light mirror to reach a photoelectric detector (photomultiplier), fluorescent signals of a light detector are converted into electric signals, the electric signals are converted into digital signals through a digital converter, lymphocyte groups are selected, lymphocytes containing cytokines such as IL-2, IFN-gamma, TNF-alpha and the like are collected through fluorescent antibody labeling, and the expression conditions of IFN-gamma, IL-2 and TNF-alpha on CD45 ⁺CD3⁺CD4⁺ and CD45 ⁺CD3⁺CD8⁺ cells are calculated through record CD45⁺CD3⁺CD4⁺、CD45⁺CD3⁺CD8⁺、CD45⁺CD3⁺CD4⁺IFN-γ⁺、CD45⁺CD3⁺CD4⁺IL-2⁺、CD45⁺CD3⁺CD4⁺TNF-α⁺、CD45⁺CD3⁺CD8⁺IFN-γ⁺、CD45⁺CD3⁺CD8⁺IL-2⁺、CD45⁺CD3⁺CD8⁺TNF-α⁺,.

The sources of reagents used in the examples of the present invention are shown in table 1 below:

table 1 example reagent information

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Collecting blank whole blood sample of rhesus monkey, namely collecting about 8.0mL of blood from jugular vein, anticoagulating with heparin sodium, and mixing the whole blood upside down before using;

PBMC extraction:

(1) And reversing and uniformly mixing heparin sodium anticoagulated whole blood.

(2) 2ML of anticoagulated whole blood was taken and added to 2mL of physiological saline (A tube) (4 sets were prepared in this ratio), and mixed well.

(3) The cells of the tube A are slowly added into a tube (tube B) containing 3mL of LSM along the tube wall, so that the liquid is spread on the LSM, 400 Xg is carried out, the temperature is 15-25 ℃, and the centrifugation is carried out for 30min.

(4) After centrifugation, the middle white cell layer was seen, the supernatant liquid was discarded, the white cell layer was transferred to a centrifuge tube (C tube) containing 3mL of 1 XPBS, mixed well, and centrifuged in a centrifuge at 250 Xg, 15-25℃for 10min.

(5) After centrifugation, the supernatant was discarded, the bottom cells were left, 1000. Mu.L of RPMI1640 complete medium was added, and the mixture was gently mixed to prepare a cell suspension.

(6) 4 Sets of PBMC were mixed and mixed uniformly, 50. Mu.L of the cell suspension was taken out, 10-fold diluted and subjected to cell counting by using a fully automatic five-class cell analyzer, and the concentration of white blood cells was detected.

(7) According to the counting result, diluting the counted cell suspension by adopting an RPMI1640 complete culture medium, so that the concentration of the cell suspension after dilution of each animal is 4-4.5X10 ⁶/mL, and culturing.

Cell stimulation and culture

(1) A negative control group, a positive control group and a sample polypeptide stimulation sample group are arranged, wherein only ProteinTransport Inhibitor (Containing BrefeldinA) is added to the negative control group, PMA, ionomycin and Protein Transport Inhibitor (Containing BrefeldinA) are added to the positive control group, and specific stimulator polypeptides and ProteinTransport Inhibitor (Containing BrefeldinA) are added to the sample polypeptide stimulation sample group.

(2) 460 Mu L of cell suspension (with the concentration of 4 multiplied by 10 ⁶/mL) after each sample is diluted is added into a corresponding sample polypeptide group pore plate;

Taking 500 mu L of cell suspension (concentration 4X 10 ⁶/mL) after dilution of each sample, and adding the cell suspension into a negative control group pore plate;

490 μl of diluted cell suspension (concentration 4×10 ⁶/mL) of randomly selected one sample per animal group was added to the positive control well plate.

(3) The sample well plate of the polypeptide group was added with 40. Mu.L of the specific stimulator polypeptide (final concentration 8.0. Mu.g/mL/well), and the positive control well plate was added with 5. Mu. LPMA dilution (final concentration 10 ng/mL/well) and 5. Mu. LIonomycin dilution (final concentration 1.0. Mu.g/mL/well), and gently mixed well.

(4) The well plate after the sample addition was placed in a carbon dioxide incubator with 37 ℃ and 5% co ₂ and saturated humidity for stimulation culture for 2 hours.

(5) After 2 hours of stimulated culture, each well plate is taken out, 1 mu L of Protein Transport Inhibitor (Containing BrefeldinA) is added into each well, the mixture is gently mixed uniformly, and the stimulated culture is continued for 12-16 hours in a carbon dioxide incubator with 37 ℃ and 5% CO ₂ and saturated humidity.

Cell collection

After the culture is completed, taking out the culture pore plate, gently blowing, uniformly mixing cells in the pore plate, collecting the cells in the pore plate into a flow tube, adding 1000 mu L of 1 XPBS, 400 Xg, 15-25 ℃, centrifuging for 5min, discarding supernatant, adding 1000 mu L of 1 XPBS, washing, 400 Xg, 15-25 ℃, centrifuging for 5min, discarding supernatant, and mixing about 100 mu L of the supernatant and the cells at the bottom of the tube into a cell suspension.

Sample processing and detection

Dead and alive dyeing

(1) 100. Mu.L of the cell suspension was aspirated separately into the flow tube.

(2) Taking 1000 mu L of diluted FixableViability Stain 780,780 in each flow tube, uniformly mixing, and incubating for 10min at room temperature in a dark place.

(3) 1000. Mu.L of 1 XPBS was added to each tube, and after washing, the mixture was centrifuged at 20℃and at 400 Xg for 5 minutes, and the supernatant was discarded.

Surface dyeing

(1) And (3) dyeing, namely respectively sucking 1.0μL BV510 MouseAnti-NHP CD45、10.0μLAPC MouseAnti-Human CD3、1.0μLBUV395 MouseAnti-Human CD4、1.0μLAlexa Fluor 700anti-human CD8a fluorescent antibodies into each flow tube, uniformly mixing, and incubating for 20min at 15-25 ℃ in a dark place.

(2) After washing, the mixture was centrifuged at 20℃with a speed of 400 Xg for 5min by adding 1000. Mu.L of 1 XPBS, and the supernatant was discarded.

Intracellular staining

(1) The reagent preparation comprises preparing Fix/PermBuffer (4×) into Fix/PermBuffer (1×) by using DiluentBuffer, preparing Perm/WashBuffer (5×) into Perm/flash Buffer (1×) by using pure water, and keeping the mixture at 2-8 ℃ in a dark place for later use.

(2) And (3) fixing and rupture of membranes, namely adding 1000 mu LTF Fix/PermBuffer (1X) into each flow tube, shaking and mixing uniformly, and incubating for 45min at 2-8 ℃ in a dark place.

(3) After washing, centrifugation was performed by adding 1000. Mu. LTF Perm/WashBuffer (1X) and washing, centrifugation conditions were 4℃at 400 Xg for 5min, and the supernatant was discarded.

(4) After washing, centrifugation was performed by adding 2000. Mu. LTF Perm/WashBuffer (1X) and washing, centrifugation conditions were 4℃at 400X 0 for 5min, and the supernatant was discarded.

(5) And (3) intracellular staining, namely adding 100 mu L TF Perm/WashBuffer (1X) into each flow tube, respectively sucking 2.5 mu L BV421 RatAnti-Human IL-2, 10.0 mu L PE MouseAnti-Human TNF-alpha and 2.5 mu L FITC MouseAnti-Human IFN-gamma fluorescent antibodies into each flow tube, shaking and mixing uniformly, and incubating at 2-8 ℃ in a dark place for 45min.

(6) After washing, centrifugation was performed by adding 2000. Mu. LTF Perm/WashBuffer (1X) and washing, centrifugation conditions were 4℃at 400 Xg for 5min, and the supernatant was discarded.

(7) And (3) sample loading and detection, namely taking out the centrifuged sample, discarding the supernatant, adding 500 mu L of 1 XPBS buffer solution for resuspension, shaking and mixing uniformly, and temporarily storing in a dark place for detection.

Data processing and analysis

Detecting a sample by using a CytoFLEX LX model flow cytometry analyzer, analyzing detection data by CytExpert software of the flow cytometry analyzer, comprehensively utilizing a histogram, a scatter diagram and a contour diagram to determine target cell groups, adjusting analysis parameters such as fluorescence compensation, gate setting position and the like as required, and confirming the distribution condition of each cell group to obtain required data. Obtain a% Parent value as a sample result

In this embodiment, a set of logical relationships is shown in FIG. 1.

The instrument parameters are shown in tables 2 and 3 below:

TABLE 2 instrument voltage, threshold

Channel name	Voltage (V)	Threshold value
			FSC	266	50000
SSC	174	/
			B525-FITC	127	/
Y585-PE	404
			R660-APC	306
R712-APCA700	616	/
			R673-APCA750	360	/
V450-PB	32
			V610	100	/
NUV450	200

Table 3 instrument compensation

The results of the tests performed using the method of the present invention, with different stimulus concentrations and different stimulus times, are shown in Table 4 below:

TABLE 4 detection results for different stimulus concentrations and different stimulus times

As a result, when PBMC was stimulated with a specific stimulator, the results showed no significant stimulation effect after 6hr of stimulation at different concentrations compared with the negative control, and after 12hr of stimulation at different concentrations of 2. Mu.g, 4. Mu.g, 8. Mu.g, the intracellular cytokine release was significant for each indicator, the results were elevated, and the 8. Mu.g concentration was the highest, consistent with the expected conditions. The detection method provided by the invention can obtain accurate results.

Experimental example

The technical scheme provided by the embodiment is subjected to methodological verification:

intra-batch Precision (WP)

The 4 samples were each tested in the same assay batch and the test was repeated 3 times. The% CV of each detection value of each sample is calculated, the% CV is 0.00-12.00% (satisfying CV less than or equal to 25%), the precision in the batch meets the requirements, and the specific results are shown in Table 5.

When the polypeptide is stimulated, a negative group and a polypeptide group are respectively carried out, 4 samples are respectively detected in the same analysis batch, and the detection is repeated for 3 times. And calculating the% CV of each detection value of each sample to obtain a negative group% CV of 0.07-34.64% (CV is less than or equal to 25% and is less than or equal to 35% by low-proportion cells), a polypeptide group% CV of 0.00-25.00% (CV is less than or equal to 25% and is less than or equal to 35% by low-proportion cells), and meeting the requirements of precision in the batch, wherein the specific results are shown in tables 6 and 7.

Batch to batch precision (Between-runPrecision, BP)

The same 3 samples were analyzed in two separate analysis batches throughout the day, each batch being tested 3 times in duplicate per sample. And calculating the% CV of each detection value of the same sample in the two analysis batches, wherein the% CV is 0.04-9.65% (the CV is less than or equal to 25%), the precision between the batches meets the requirements, and the specific results are shown in Table 8.

TABLE 5 within-batch precision of T lymphocyte subtype intracellular factor detection protocol in rhesus peripheral blood

TABLE 6 within-batch precision of detection of negative samples for T lymphocyte subtype intracellular factor in rhesus peripheral blood

TABLE 7 within-batch precision of T lymphocyte subtype intracellular factor detection positive samples in rhesus peripheral blood

TABLE 8 batch-to-batch precision of T lymphocyte subtype intracellular factors in rhesus peripheral blood

The verification result shows that the precision CV range in the batch is 0.24-14.21%, the precision CV range between batches is 0.03-16.03%, and when the polypeptide is stimulated, the negative group and the polypeptide group are respectively carried out, 4 samples are respectively detected in the same analysis batch, and the detection is repeated for 3 times. Calculating the% CV of each detection value of each sample to obtain a negative group% CV of 0.07-34.64% (satisfying CV less than or equal to 25%, satisfying CV less than or equal to 35% for low proportion cells), a polypeptide group% CV of 0.00-25.00% (satisfying CV less than or equal to 25%, satisfying CV less than or equal to 35% for low proportion cells), and meeting the requirements of in-batch precision, so that the method has accurate detection results and can be used for detecting intracellular factors of T lymphocyte subtypes in peripheral blood of rhesus monkeys.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A method for detecting intracellular factors of a rhesus T lymphocyte subtype, comprising the steps of:

sample processing including dead and alive staining, surface staining, cell rupture and intracellular staining;

the surface staining comprises the steps of taking antibody combinations 1 with different fluorescent labels, and mixing and incubating with a sample to be detected;

detecting through flow cytometry, and obtaining detection data results of the macaque T lymphocyte subtype intracellular factors;

The antibody combination 1 comprises a CD45 fluorescent antibody, a CD3 fluorescent antibody, a CD4 fluorescent antibody and a CD8a fluorescent antibody.

2. The method of claim 1, wherein the fluorescent label of the CD45 fluorescent antibody is BV510;

the fluorescent label of the CD3 fluorescent antibody is APC;

the fluorescent label of the CD4 fluorescent antibody is BUV395;

the fluorescent label of the CD8a fluorescent antibody is AlexaFluor 700.

3. The detection method according to claim 2, wherein the volume ratio of the CD45 fluorescent antibody, the CD3 fluorescent antibody, the CD4 fluorescent antibody and the CD8a fluorescent antibody is 0.5-2:8-12:0.5-2:0.5-2.

4. The method of claim 1, wherein the dead-alive staining is Fixable Viability Stain 780,780.

5. The method according to claim 1, wherein the cell disruption is TF Fix/Perm Buffer.

6. The method according to claim 7, wherein the intracellular staining comprises incubating a combination of antibodies 2 labeled with different fluorescent markers in combination with the sample to be detected;

the antibody combination 2 comprises IL-2 fluorescent antibody, TNF-alpha fluorescent antibody and IFN-gamma fluorescent antibody.

7. The method according to claim 6, wherein the fluorescent label of the IL-2 fluorescent antibody is BV421;

The fluorescent label of the TNF-alpha fluorescent antibody is PE;

the fluorescent label of the IFN-gamma fluorescent antibody is FITC.

8. The method according to claim 6, wherein the volume ratio of the IL-2 fluorescent antibody, the TNF-alpha fluorescent antibody and the IFN-gamma fluorescent antibody is 0.5-1.5:3-5:0.5-1.5.

9. The method of claim 1, wherein the obtained test data includes data acquisition and data analysis;

The data analysis includes selection CD45⁺CD3⁺CD4⁺、CD45⁺CD3⁺CD8⁺、CD45⁺CD3⁺CD4⁺IFN-γ⁺、CD45⁺CD3⁺CD4⁺IL-2⁺、CD45⁺CD3⁺CD4⁺TNF-α⁺、CD45⁺CD3⁺CD8⁺IFN-γ⁺、CD45⁺CD3⁺CD8⁺IL-2⁺、CD45⁺CD3⁺CD8⁺TNF-α⁺ of subpopulation expression profiles.

10. The method according to any one of claims 1 to 9, wherein the sample is derived from rhesus peripheral blood.