The stimulation time you are using is enough to induce IFNg and IL-17 production in splenocytes and IL-10 in the lung infiltrating cells.

Check the antibody you are using, does they work well

Also what are the cells you are looking at, they might be conventional CD4s or Tregs

Thanks. I forgot to mention these are from naïve mice. The cells are from lung homogenates made into single cells and splenocytes. So theya re a mixture of all kinds of immune cells, however I am gating on CD3+Cd4+ cells to look for IFNg, Il-13 and so on for cytokine detection. And since these are naïve mice, I was wondering if I should stimulate them longer like 18-24hrs to detect cytokines?

Thanks!

You should be able to observe IFNg and IL17 positive CD4 cells atleast in spleen, if it is naive BL6 mice.

The 4hr stimulation is enough for this stimulation. However if you are using anti-CD3/CD28 mAb then 18-24hrs is best.

The 4 hr stimualtion with PMA and Ionomycin is inducing significant cell death, if you prolong it you might have less cells to analyze the data

I see. I am not using anti-CD3/Cd28 for this. I am going to try this again and find out if that works. Thanks a lot for your suggestion.