I have aliquots of expanded cytotoxic T-cells that I would like to use directly in a cytoxicity assay after thawing the cells without re-stimulating them.
So far I've tried thawing them, resting them overnight in IL-2, and then using them in experiments but gotten no response. The viability of the thawed cells was excellent, they just weren't cytotoxically active. Does anybody else do this routinely? What can I do to recover the cytotoxicity of the cells after thawing them? My current thought is just to rest them longer (like 48 hours) - is it as simple as that or am I missing something else?
I had experience working on Cr-labeled cytotoxic assay but I always used fresh T-lymphocytes. Cromium is supposed to be released when the labeled cells are dead. That is the criteria of cytotoxicity. While I m not asking your target cells, I do have the question of vitality of your T cells. Are you sure they were alive?
Have you tried eosin dye to check on the vitality of cells involved? Also, I do not know how long the T cell had been kept frozen (in liquid nitrogen I presumed?). If you had frozen cells for a long time, you should expect portion of cells would be lost (they have mix of ages) . Also, there is a possibility, if the T cells were frozen for a long time, water would enter cell membranes and formed crystal deposit. So, you should give more time for T cells to recover before you work on cytotoxicity assay.
Shih-Wen Huang, MD
I'm sure they are still alive. I checked them using trypan + automated cell counter as well as loading with propidium iodide and measuring in flow cytometry. Just by visual inspection, their morphology looked great also. I didn't use any metabolic stains like DTT, that may be something to consider for future troubleshooting. The cells had been stored in liquid nitrogen for less than a month.
If you are confident all your frozen cytotoxic T-cells are alive and well, you should start asking question on your target cells.
Shih-Wen Huang, MD
- Badges
- Science topic