Cytotoxicity Assay Protocol

Begin T cell culturing a week prior. Grow and culture tumor cell lines. Ensure the cells are not over-confluent. 

Split the tumor cells the day before the assay. Perform the cytotoxic assay as described in next steps.

Count tumor cells. Collect 3-5M cells for each cell line.

Centrifuge the cells and wash the pellet once with PBS. Ensure that all media is washed off as this can inhibit the staining.

Aspirate the supernatant and leave as little PBS as possible

Re-suspend cells in culture medium at the appropriate ratio for the experiment (1X10⁶ / ml for 50,000

cells/50ul/well or about ~80% confluent) using 96 well round well plate with 50,000 cell tumor and in triplication.

Add 50 ul of T cell suspension diluted to achieve the desired E:T ratio. Below is an example of how to set up E:T ratios:

A	B	C
ET Ratio(s)	T cells/50ul /well	Tumor cells/50ul/well
0:1	0 (only with T cell medium)	5 x104 cells
1:1	5 x104 cells	5 x104 cells
3:1	1.5x105 cells	5 x104 cells

Centrifuge the mixed cells in the tubes at 300 rpm for 5 minutes to gently pellet the cells. Incubate for 3 hours at 37⁰C.

Add 150ul PBS into the plate and then centrifuge at 2000rpm for 5min.

Remove the supernatant and add then 100ul/well Trypsin. The plate is incubated at 37C incubator for 3-5mins.

Add 100ul FACS buffer to stop the trypsinization and centrifuge at 2000rpm for 5 min.

Remove the supernatant carefully.

Fix and permeabilize cells using Cytofix/Cytoperm^TM Kit (BD BioSciences) for 20 minutes at room temperature (100ul/well – mix well ).

Wash with Perm/Wash^TMBuffer (150ul/well) at 2000 rpm for 5 min.

Prepare caspase antibody dilution such that each well gets stained with 2.5ul of APC-conjugated rabbit anti-active caspase 3 Ab in 50 ul Perm/Wash^TM Buffer. Mix gently and incubate for 30 min in the dark on ice or in the 4°C refrigerator.

Wash cells 1Xwith Perm/Wash^TMBuffer (150ul).

Resuspend the cell pallets into 100ul FACS buffer (2% FBS in 1X PBS).

Analyze the samples using flow cytometry.