I'm stimulating CD4+ T cells for 3 and 5 days with plate-bound CD3 antibody, soluble CD28 antibody, and LPS. I want to measure intracellular cytokine levels on days 3 and 5 (IL4, IFNγ, IL10, and IL17A) via intracellular staining and flow cytometry. When should I add brefeldin A to my cells in order to detect intracellular cytokines? 4 hours before analysis? 12 hours? Any help would be appreciated.
P.S. I've already analyzed T cells stimulated for 3 days for IL17A. I added brefeldin A 4 hours before analyzing the cells, but I didn't see any IL17A+ T cells. This is making me worry that perhaps I didn't add BFA add the optimal timepoint.
I add BrfdA at 5 μg/mL final, together with re-stimulating peptide, and incubate for 4 hr before harvest, for my activated and IL2-expanded OT-1 CD8 T cells, and detected nicely Ifng and Tnf.
Do your cells require re-stimulation to produce IL17A? Was this done?
Thomas,
That is a great question. I'll have to try restimulation. I just assumed that since the cells are cultured with plate-bound CD3e antibody, then the cells should just be constantly stimulated. But maybe restimulating PMA/Ionomycin would lead to IL17A+ cells. That's something I need to look into.
A constant stimulation means death to T cells, normally by 48h of simulation I would remove the TCR simulation and resuspended cells in a fresh culture with IL2 to expand them for about 2 days. These cells will grow in number but won't produce effector cytokines until they are re-stimulated. This applies to CD8 cells but CD4 should be very similar apart from some different cytokines you would use to differentiate them.
Thomas Ching-Jen Tan This is exactly the kind of information I was looking for. Thank you! I'll try stimulating for 48 hours and then restimulating my CD4 T cells after their expansion.
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