Hello everyone,
I am working on T cell proliferation assay using 3H-thymidine. I am using PBMC to culture for 3 days (72 hourse). I am using IL-2 for T cell activation. Can any please tell me, whether it is possible to treat the cell with IL-2 & 3H-thymidine on the same day with a suitable protocol. Thank you in advance.
Best Regard
Razaul Haque
IL-2 will not activate T cells, it would just sustain proliferating T cells. You add it to your culture in the beginning to make sure that your activated T cells will keep proliferating and not die. To activate T cells, you need to use a stimulant (eg. aCD3/CD28) and you need to wait for 3-6 days to see optimal proliferation (which you are trying to measure by thymidine incorporation). You could wait and add IL-2 along with thymidine towards the end of your culture but this won't be ideal as you won't see much proliferation. You could do this early in your culture but again, you won't see much proliferation. Long answer short, you could do it but your results won't be great.
Dear Julie Joseph,
Thank you so much for your kind suggestion. I will follow your suggestion.
Dear Razaul, Julie is right saying that IL-2 itself will not activate T cells (only very high concentrations can do it). Evidently, T cells need to obtain first signal for activation, which can be provided by HLA/peptide complex specific to TCR, mAbs to CD3 or any plant lectin substances, such as phytohemagglutinin (PHA, 5-10 mkg/ml) or concanavalin A (Con A, 2-5 mkg/ml). Second signal for proliferation may be costimulatory (anti-CD28) or cytokine (IL-2). Obviously, both signals should be provided at the start of T cell culturing.
Dear Dmitry B. Kazansky,
Thank you for your kind suggestion. I am culturing PBMC for 3 days (72 hours). On day 0: I usually start culturing by stimulating with CD3. On day 2: I treat the cells with IL-2. On day 3: I harvest the cells after incubating 18 hours with 3H-thymidine incorporation. My query is whether it is possible to treat the cells with IL-2 and Thymidine on the same day ( on day 2) or not. If you please give me a suggestion regarding this I will be helpful.
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