CN114561353A - Immunological method for efficiently separating and purifying NKT cells - Google Patents

Immunological method for efficiently separating and purifying NKT cells Download PDF

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CN114561353A
CN114561353A CN202210317128.5A CN202210317128A CN114561353A CN 114561353 A CN114561353 A CN 114561353A CN 202210317128 A CN202210317128 A CN 202210317128A CN 114561353 A CN114561353 A CN 114561353A
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邹瑶
王婷婷
于超
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Chongqing Medical University
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Abstract

The invention provides a method for efficiently separating and purifying NKT cells, and relates to the technical fields of immunology, molecular biology, molecular immunology and the like. The method for separating and purifying comprises the following steps: 1) sorting T cells; 2) sorting NKT cells; 3) verification of purity identification of NKT cells; 4) selection of the number of spleen cells in the sorting; the invention obtains the high-purity NKT cells, and has far-reaching significance in the aspect of cellular immunotherapy of cardiovascular diseases, tumors and autoimmune diseases for the NKT cells.

Description

Immunological method for efficiently separating and purifying NKT cells
Technical Field
The present invention relates to the fields of immunology, molecular biology, molecular immunology, etc. The method specifically comprises sorting of T cells, sorting of NKT cells and purity identification of NKT cells.
Background
Natural killer T cells (NKTs) play an important role as a specific subset of T cells in both innate and adaptive immunity. In mice, NKT cells are defined as CD3+ NK1.1+ NKT; NKT cells are defined as CD3+ CD56+ NKT in humans. NKT cells express both T Cell Receptors (TCR) and NK cell receptors. NKT cells can promote cell-mediated antitumor and anti-infection effects, and can prevent autoimmune diseases by regulating immune tolerance. As a bridge between innate and adaptive immunity, NKT cells function under a variety of pathological conditions. NKT cells currently play their role primarily by means of 3 pathways: 1. after antigen activation, NKT cells can exert effects by secreting cytokines such as IFN-gamma, IL-4, IL-10 and the like; after NKT cells are activated, perforin and granzyme can be released; 3. activated NKT cells, the release of relevant cytokines, activate neighboring NK cells, B cells and antigen presenting cells. When NKT cells are activated, they express a large number of cytokines, including proinflammatory factors such as IFN γ, IL-15, IL-18 and TNF α, and anti-inflammatory factors such as IL-4, IL-10 and IL-13. On one hand, the cytokines can directly act on NKT cells to play a cytotoxic role, and on the other hand, the cytokines can transactivate other immune cells such as natural killer T cells, B cells and the like to play an immunoregulation role. It is therefore widely believed that NKT cells mediate protective and regulatory immune functions, playing a regulatory role in tumors, infections and autoimmune diseases.
Nowadays, cellular immunotherapy is vigorously developed. As a new biological treatment method, the cellular immunotherapy method refers to the method of effectively regulating and controlling the immune balance of the organism and recovering the functional disorder of the immune system of the organism by adoptively infusing autologous cells induced and amplified in vitro. The international cell transfer therapy provides a very important research idea for treating various diseases caused by the imbalance of an immune cell system, such as tumors, autoimmune diseases and cardiovascular diseases. Therefore, it is important to obtain specific functional immune cells for the study of cellular immunotherapy.
CD3+CD56+NKT cells, as a novel cytotoxic effector cell, have the advantages of strong immunocompetence of T lymphocytes and non-MHC (major histocompatibility complex) limited broad-spectrum immunity of NK cells, and have small influence on a bone marrow hematopoietic system, are cells commonly used in current adoptive immunotherapy of blood system tumors, autoimmune diseases and cardiovascular diseases, and are considered to be the first choice for adoptive cellular immunotherapy. However, the isolation and purification of NKT cells still have great difficulty at present.
Therefore, the method for efficiently separating and purifying NKT has important significance for treating autoimmune diseases, tumors and cardiovascular diseases.
Disclosure of Invention
The invention provides a method for efficiently separating and purifying NKT cells, which comprises the following specific steps:
1. separation and purification of T cells in mouse spleen:
1) separation and purification of T cells in spleen of C57 mouse
(1) C57 mice were sacrificed after anaesthesia with chloral hydrate and soaked in 75% ethanol for several minutes.
(2) Dissecting the mouse, taking out the spleen, placing the spleen on a steel mesh, adding a PBS solution, and grinding.
(3) After milling, spleen cell suspension was collected in 15mL centrifuge tubes.
(4) The centrifugation operation is carried out at room temperature and 1800rpm for 5 min.
(5) After centrifugation, the supernatant was discarded, 3mL of erythrocyte lysate was added, and the suspension was resuspended.
(6) After 5 minutes of lysis, centrifugation was carried out at 1800rpm for 5 minutes at 1500-.
(7) After centrifugation, the supernatant was discarded, and 3mL of erythrocyte lysate was added again and resuspended.
(8) After 5 minutes of lysis, centrifugation was carried out at 1800rpm for 5 minutes at 1500-.
(9) After centrifugation, the supernatant was discarded, and 3ml of LPBS solution was added and resuspended.
(10) The centrifugation was carried out at 1500 ℃ and 1800rpm for 5 min.
(11) After centrifugation, the supernatant was discarded, and 1ml of LPBS solution was added and resuspended.
(12) The above-mentioned partial spleen cell suspension was counted, and the remaining cell suspension was centrifuged at 1800rpm for 5min at the same time.
(13) Adjusting the cell number, and taking 0.1-2mL of cell suspension.
(14) Rat serum (50ul/ml) was added and resuspended.
(15) The liquid was transferred to a 5ml bottom round polypropylene tube and EasySep was addedTMMouse T Cell Isolation Cocktail (50ul/ml), resuspended, and incubated at room temperature for 10 min.
(16) Vortex EasySepTM Dextran RapidSpheresTM，30s。
(17) EasySep is added into the test tubeTM Dextran RapidSpheresTM(75ul/ml), resuspended, and incubated at room temperature for 2.5 min.
(18) The tubes were filled to 2.5ml with medium (PBS + EDTA + FBS) and resuspended (2-3 puffs).
(19) The tubes were placed in a magnet and incubated at room temperature for 2.5 min.
(20) Pouring is rapidly finished at one time, and the poured solution is the T cell suspension.
2)Apoe-/-Separation and purification of T cells in mouse spleen
Experimental procedures same as 1) separation and purification of T cells from spleen of C57 mouse
2. Isolation and purification of NKT cells from mouse T cell suspension
1) Isolation and purification of NKT cells from C57 mouse T cell suspension
(1) Sorting with the above obtained T cell suspension, adjusting cell number, and controlling at 108cells/mL suspension;
(2) taking 0.5-2mL of the suspension, transferring to 5mL of a polypropylene round bottom tube, and adding EasySepTMMouse NK Cell Isolation Cocktail (50ul/ml), resuspended, and incubated at room temperature for 10 min;
(3) vortex easy SepTM Dextran RapidSpheresTM，30s；
(4) EasySep is added into the test tubeTM Dextran RapidSpheresTM(100ul/ml), resuspending, and incubating at room temperature for 5 min; adding the culture medium (PBS + EDTA + FBS) to 2.5ml, and resuspending (2-3 times by air-blowing);
(5) the tube (without lid) was incubated in a magnet at room temperature for 5 min;
(6) picking up the magnet, rapidly pouring at one time, and obtaining a suspension after pouring;
(7) the tube in the magnet was aspirated, medium (PBS + EDTA + FBS) was added to 2.5ml, and resuspended (5-6 puffs);
(8) placing the test tube (ensuring the test tube is dry) in a magnet and incubating for 5min at room temperature;
(9) and (4) picking up the magnet, quickly pouring at one time, and pouring to obtain a suspension (mixing the suspension with the suspension obtained in the step 6), so as to obtain the NKT cells.
2)Apoe-/-Isolation and purification of NKT cells from mouse T cell suspension
Experimental procedures same as 1) isolation and purification of NKT cells from the suspension of mouse T cells C57
3. Selection of spleen cell number
1) When the number of spleen cells is more than or equal to 108cells, separating and purifying spleen cells according to the steps and detecting the purity.
2) When the number of spleen cells is less than or equal to 5x107cells, separating and purifying the spleen cells according to the steps, and detecting the purity of the spleen cells.
Drawings
FIG. 1 is a simplified flow chart of NKT cell sorting and purification
FIG. 2 is a flow cytometric curtailment of NKT cells
FIG. 3 is a graph showing the results of the isolation and purification of C57 mouse NKT cells.
FIG. 4 is Apoe-/-Figure of the results of mouse NKT cell isolation and purification.
FIG. 5 shows spleen cell number ≥ 108cell, NKT cell isolation and purification.
FIG. 6 shows that the number of spleen cells was ≦ 5 × 107cell, NKT cell isolation and purification.
Detailed Description
The apparatus used in the following examples of the invention is as follows:
(flow cytometer, Beckman, USA),
the embodiments of the present invention are described in further detail below:
example 1: the NKT cell purity identification method comprises the following specific steps
Purification and identification of NKT cells
1) Purity characterization of C57 mouse NKT cells
(1) Transfer 100-120. mu. LNKT cell suspension into 96-well plates.
(2) The centrifugation was carried out at 1500 ℃ and 1800rpm for 5 min.
(3) Preparing a flow-type staining antibody: Anti-mica-PEcy 7-CD45 flow antibody, Anti-mica-FITC-CD 3 flow antibody and Anti-mica-BV 650-NK1.1 flow antibody.
(4) After completion of centrifugation, the supernatant was discarded, 50. mu.L of the prepared flow antibody was added, and resuspended.
(5) Incubated at 4 ℃ for 30-40 minutes in the absence of light.
(6) After completion of incubation, 100 and 150. mu.L of PBS solution/stabilizing buffer (FBS: PBS 1:100) was added and resuspended.
(7) The centrifugation was carried out at 1500 ℃ and 1800rpm for 5 min.
(8) After completion of the centrifugation, the supernatant was discarded, and 100. mu.L of PBS solution was added and resuspended.
(9) Transfer to 1.5ml lep tube, ready for detection on flow machine.
2)Apoe-/-Purity characterization of mouse NKT cells
Experimental procedures same as 1) purity identification of C57 mouse NKT cells
FIG. 3 is a graph showing the isolation and purification results of mouse NKT cells from C57, which shows that high-purity NKT cells can be isolated from C57 mouse spleen cells, and the purity is as high as 95%.
FIG. 4 Apoe-/-The results of the isolation and purification of mouse NKT cells are shown in the figure, and the results show that Apoe can be extracted from the cells-/-High-purity NKT cells are separated from mouse spleen cells, and the purity is as high as 95%.
FIG. 5 spleen cell number ≥ 108In cells, it was found that the NKT cells isolated from mouse spleen cells were not significantly grouped and that the NKT cells were of low purity.
FIG. 6 spleen cell number ≤ 5 × 107The cell and NKT cell separation and purification map shows that the cell groups separated from mouse spleen cells are obvious, the cell viability is high, and the NKT cell purity is as high as 95%.

Claims (5)

1. A method for efficiently separating and purifying NKT immunological cells comprises the following steps:
(1) separating and purifying T cells;
(2) separating and purifying NKT cells;
(3) identification of NKT cells;
(4) selection of spleen cell number in sorting.
2. The method of claim 1, wherein the T cell separation and purification comprises the following steps: at room temperature, the serum is collected from rat and EasySepTMMouse T Cell Isolation Cocktail、EasySepTMDextran RapidSpheresTMAnd (4) treating, putting into a magnet for incubation, and pouring the obtained liquid to obtain the T cell suspension.
3. The method of claim 1, wherein the NKT cells are isolated and purified by the steps of: the T cell suspension obtained above was subjected to EasySep at room temperatureTMMouse NK Cell Isolation Cocktail、EasySepTMDextran RapidSpheresTMTreating, putting a magnet for incubation, and pouring the obtained liquid to obtain NKT cell suspension; adding the culture medium again for resuspension, putting a magnet for incubation, and pouring the obtained liquid to obtain NKT cell suspension; and combining the two liquids to obtain the separated and purified NKT cell suspension.
4. The method of claim 1, wherein the obtained NKT cells are identified by flow-staining, and the obtained NKT cells are high in expression of CD3 and NK1.1 and have purity of up to 95%.
5. The method of claim 1, wherein the spleen cell number is selected by the following steps: selecting spleen cells with different numbers for sorting according to the method 2, when the number of the spleen cells is more than or equal to 108In cells, it can be seen that the cells isolated from mouse spleen cells are not well grouped, and NKT cells have low purity; when the number of spleen cells is less than or equal to 5x107cells can be seen from the figure, the cell groups separated from the mouse spleen cells are obvious, the cell survival rate is high, and the NKT cell purity is as high as 95%.
CN202210317128.5A 2022-03-28 2022-03-28 Immunological method for efficiently separating and purifying NKT cells Pending CN114561353A (en)

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