Functional assay of cytotoxic T cells induced by specific antigens

Summary

Specific antigen-induced cytotoxic T cell function assay is mainly applied to (1) the determination of immune cell function (2) the diagnosis of clinical autoimmune diseases (3) polyclonal secondary signal transduction molecules.

Operation method

Functional assay of cytotoxic T cells induced by specific antigens

Principle

In this method, antigen-specific CTLs are first obtained with the help of long-term mixed lymphocyte cultures and then subjected to cytotoxicity testing. The principle is that peripheral blood lymphocytes contain CTL clones specific for different antigens, and after in vitro stimulation with a specific (or allogeneic) antigen, the T cell clones that can recognize the antigen are selectively activated and proliferate, whereas the other T cell clones gradually die; after 3-4 stimulations, the surviving ones are the ones that recognize the specific MHC/antigenic peptide complex, i.e., the antigen specific CTL. CTL .

Materials and Instruments

B lymphoblastoid cell line Blood sample
Mitomycin C PBS RPMI 1640 medium
24-well culture plate Centrifuge Incubator

Move

1. Induction and preparation of specific CTLs

① Take the author's peripheral blood isolated PBMC, serum-free 1640 washed twice, with RPMI 1640 containing 20% of neonatal bovine serum to 1.5 × 106 /ml, placed in 24-well plates, in 5% CO2 incubator for 4 hours to make the monocytes affixed to the wall in order to remove it, and then the cells were collected and counted;

② Take EBV-transformed B lymphoblastoid cells, add mitomycin C at a final concentration of 30 μg/ml, act in a 37℃ water bath for 30 min, centrifuge at 1000 r/min for 10 min, discard the supernatant, and the precipitated cells were washed with 1640 solution for 3 times and counted;

③ Take 2×106 PBL in a 24-well plate, add 5×104 (2.5%) self, allogeneic (their HLA-I types are completely different) EBV-LCL cells treated with mitomycin C (30mg/ml, 30min) as the stimulating cells, mix well, and replenish the total volume to 2 ml with complete medium (RPMI 1640);

④ Static placed in the incubator; after 4d half volume change, continue to culture for 3d;

⑤ Collect cells by centrifugation, take 1×106 response cells, add 2×105 (20%) of stimulated cells, and add recombinant IL-2 on the third day to make the final concentration of 30U/ml; change the fluid in half every three days and maintain the same IL-2 concentration.

⑥ Stimulate the effector cells once a week according to the same procedure; after 3~4 times, the effector cells are specific CTL and can be used for killing experiments.

2. Cytotoxicity test

Detection of CTL cytotoxicity can be used to detect NK cell killing activity, only the proportion of effector target cells are not the same, now the LDH release method is briefly described as follows:

① Target cells are self or allogeneic EBV-LCL cells used as stimulating cells, adjusted to 1×105/ml;

② The effector cell is the specific CTL induced as described above, adjusted to 2.5×106/ml;

③ Take 0.1 ml of each in a 96-well plate (the ratio of effector/target is 25:1); blow gently to mix the two; and set up a target cell natural release control group (i.e., only add target cells without effector cells) and a maximum release control group (0.1 ml of target cells and 0.1 ml of 1% NP-40);

④ After centrifugation at 1000rpm/min for 2min, incubate at 37°C in a 5% CO2 incubator for 4hr.

⑤ Enzymatic color reaction: see "NK cell killing assay".

Caveat

1. Induction and preparation of the cells to enable the removal of mononuclear cells from the wall before starting to collect the cells to prevent cell contamination.

2. Set up control groups to prevent false-positive or false-negative results.

3. Biological contamination can lead to a decrease in cell proliferation.

Common Problems

1. When using frozen cells, the viability of the cells needs to be tested after recovery.

2. serum added to the culture medium, some serum may inhibit the reaction.

3. background should be less than 2000 rpm, negative controls should be greater than 20,000 rpm.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/