CD8+ T cells, NK, NKT cells were stimulated by anti CD3, CD28 for 1 hour, 4 hours, 24 hours to produce cytokines. Then, brefeldin A was added after stimulation times to keep cytokines inside cells, and they analysed by flow cytometry. However, there was no cytokines expression in all time course. Is any one have any suggestion or explanation???
Using this stimulation condition, the time frame used is not adequate to induce cytokine expression.
I don't think the problem would be with the stimulants but the brefeldin A is the one which blocks the secretion of cytokines and you have to reconsider if you use an appropriate concentration. Your fixation and permeabilization steps are also critical. This technique has multi steps and it is hard to point out where the problem is but you need to optimize each step. May be if there is a problem with the stimulants or the cytokines accomulator , you can check the secretion of the cytokine from the supernatant.Good luck.
Hi Mohammed,
Which cytokines are you trying to measure? Is this a stimulation of naïve cells or a re-stimulation of already activated cells? For naïve T cells, 3+ days would be a better time point. As a re-stimulation, 4 hours is sufficient. And Brefeldin A should be in your cell culture for the last 4-6 hrs of stimulation.
Good Luck!
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