Specific antigen-induced cytotoxic T cell function assay
Experimental reagent
Mitomycin C (Sigma): 300 μg/ml in culture medium or PBS
RPMI 1640 with 20% newborn calf serum
Experimental Materials
Epstein-Barr virus-transformed B lymphoblastoid cell line
Experimental procedure
1. Induction and preparation of specific CTL
1) PBMC was isolated from the peripheral blood of the author, washed twice with serum-free 1640, adjusted to 1.5 × 10 6 /ml with RPMI 1640 containing 20% ​​newborn calf serum, placed in a 24-well plate, in a 5% CO 2 incubator The monocytes were attached to the wall for removal in 4 hours, and then the cells were collected and counted;
2) Take EB virus-transformed B lymphoblasts, add mitomycin C, the final concentration is 30μg/ml, apply in water bath for 37min at 37°C, centrifuge for 10min at 1000r/min, discard the supernatant, and wash the cells with 1640 solution. 3 times and count;
3) Take 2 × 10 6 PBLs in a 24-well plate, add 5 × 10 4 (2.5%) of mitomycin C (30 mg / ml, 30 min) of self, allogeneic (HLA-I EBV-LCL cells of different types are used as stimulating cells, mixed, and the total volume (RPMI 1640) is used to make up the total volume to 2 ml;
4) Statically placed in the incubator; after 4 days, the amount of liquid change, continue to culture for 3d;
5) Collect the cells by centrifugation, take 1 × 10 6 reaction cells, add 2 × 10 5 (20%) of the stimulated cells, and add recombinant IL-2 on the third day to a final concentration of 30 U/ml; Change the solution once and maintain the same IL-2 concentration.
6) The effector cells are stimulated once a week by the same procedure. After 3 to 4 times, the effector cells are specific CTLs, which can be used for killing experiments.
2. Cytotoxicity test
The detection of cytotoxicity of CTL can be carried out by detecting the killing activity of NK cells. The ratio of target-only cells is different. The LDH release method is briefly described as follows:
1) The target cells are self- or allogeneic EBV-LCL cells used as stimulator cells, adjusted to 1 × 10 5 /ml;
2) The effector cells are the specific CTL induced above, adjusted to 2.5×10 6 /ml;
3) Take 0.1ml each in a 96-well plate (effect/target ratio is 25:1); gently blow to mix the two; at the same time set the target cells to naturally release the control group (ie, only add target cells without effect) Cells) and the maximum release control group (0.1 ml target cells and 0.1 ml 1% NP-40);
4) After centrifugation at 1000 rpm/min for 2 min, incubate in a 37 ° C, 5% CO 2 incubator for 4 hr;
5) Enzymatic color reaction: See "NK cell killing test".
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