Hi, I am currently design a assay for testing the cytotoxicity of primary T cells against tumor cell lines. Here is the design:
Cryo-preserved PBMCs were retrieved and cultured with 9ug/mL PHA, 20%FBS RPMI1640 medium for 48 hours. Then the PBMCs were stained with CD3 antibody and were sorted for CD3+ T cells. The adherent tumor cell lines A549 were seeded the day before add T cells in 96 well plate (3000 cells/well). Tumor cells were stained with LIVE/DEAD® Viability/Cytotoxicity Kit (Themo Fisher: L3224). After T cell purification and tumor cell staining, we add T cells to tumor cells in 200 ul medium. Finally, the cytotoxicity were observed in In-Cell-Analyzer 2200 (GE) for 6 hours by real-time imaging and count for red signal of dead tumor cells. The RNA of T cells were collected after recording of images, and cytotoxicity genes expression, like IFNG, PRF1, GZMB, will be run on real-time qPCR. CD3 will be used as housekeeping gene.
I know the PHA will activate T cells and stimulate them proliferating via CD2 and calcium influx pathway. And I do observe obvious cytotoxicity in the preliminary experiments. Could anybody give me some advices to improve this method? .
Hi, Ying, our target cell is adherent cells, therefore there is not possible to use flow, E:T is 10:1, we has 3000 cells in a well.
In our experience PBMCs do not survive well after cryopreservation. However, when we did T cell cytotoxicity against tumor cell lines, we probed our cells with radioisotopes.
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