We want to measure the level of activation of the different populations of CD3 positive cells after a CD3 sorting (using FACS Aria, CD3-PB). We stimulate the cells obtained from the sorting with a coating of anti-CD3 and anti-CD28, 3 hours before the seeding. Maintaining the stimuli for 72 hours renders a spectacular decrease in the fluorescence intensity for CD8 molecule, and the cells seems to be CD8 negative or dim (CD4 cells are not affected) . Does anyone have the same results? Which could be the possible explanation? Thanks in advance!
Why do you need to cultivate T cells so long? Obviously, activation events are measured shortly after activation (minutes, hours, or 1-2 days maximum). Possible explanation of your effect is that CD8 T cells are weak producers of IL-2. They proliferate more quickly as compared with CD T cells and begin competition for IL-2. So, 3 days later you can see apoptotic death due to deprivation of this cytokine.
Thank you Dmitry! We are trying different durations in order to optimize a co-culture experiment, so we can have a broader window of action. We will think about your answer!
It may be, you will need to add external IL-2 to cultures containing CD8+ T cells. I think, 5-10 IU/ml will be sufficient. Alternative approach would be to limit incubation time by no more than 24 h.
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