Hi, guys. I want to use MACS-sorted mouse CD4+ T cells from spleen and LNs to test the effect of an drug and some directors asked me to culture T cells without IL-2/CD3/28 to exclude the effect of these stimulation. However, I cultured it and find that cells under 16h culture broken into pieces after Foxp3 nuclear staining fixation when I am using flow cytometry for analysis. May I know how long (how many hours) can I keep MACS-sorted CD4 T cells without IL-2 in RPMI1640? I found if the culture time is too long cells will die or break into pieces after Foxp3 staining fixation, such as 24h. But how long is enough without killing them? May I know your experience in T cells culture? Someone tell me that they can ensure 50% of CD4+ T cells alive at 24h through reduce the medium volume and increasing the cell density. However, may I reply the director that too many cells under cell death at 16h and 24h and limited CD4Foxp3 Treg ratio analysis?
Thanks
Did you check whether you cells were dead before or after intracellular staining? Like your colleague suggested, you could try plating lots of cells in a smaller volume/surface area and see whether you can reduce cell death.
It is standard practice to add aCD3/CD8 + IL-2 when you put T cells in culture. In their absence, T cells tend to die off. If your director really wants to avoid adding aCD3/CD28, you could maybe add your drug + IL-2. If you still see lots of cell death, you might have no choice but to add your drug to T cells plated with aCD3/CD28/IL-2. You could then normalize the response you see to the cells that were plated with aCD3/CD28/IL-2 but no drug.
Hi Shuoyang,
T cells will have poor viability even after 24 hours if not adequately supplied with IL-2 (concentration dependent upon cell type) and aCD3/28 (1 ug/ml for effectors, typically). Without either of these, the T cells will begin to undergo apoptosis. You can see this very clearly at later time points, by cell culture media not changing colour in comparison to stimulated wells and other visual clues i.e. no T cell clumping, as is common with activated CD4+'s.
I would suggest 3 possible solutions:
1. Use suboptimal IL-2 concentration; enough to ensure 'some' T cell survival. However, be careful of any conclusions you may draw from such strained settings.
2. Use IL-7 to promote cell survival. The catch is that is should be added often and a constant concentration maintained. This maintenance is important as at high concentrations IL-7 promotes proliferation, whilst low dose boosts survival.
3. Use an aCD3/CD28 and IL-2 supplemented well, an unstimulated/no IL-2 control and aCD3/CD28/IL-2/drug well to demonstrate initially how essential each of these are to your cultures, and then normalise your +drug well to -drug well, to account for its effects (as Julie mentioned above).
Good luck!
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