I want to know by FACS if a population of cells express several cytokines in basal conditions (no activation) within a given tissue. I directly get those cells ex-vivo from human tissue samples, so I fear I may not to be able to detect the cytokine because no inhibition of cytokine secretion can be applied to a human tissue sample, obviously. Can anyone suggest a protocol for intracellular cytokine staining on ex-vivo cells? Is it necessary to treat/culture ex-vivo cells with brefeldin A prior staining, or can a regular intracelular staining with PFA/saponin be made directly after cell isolation?
Hi Maria,
in order to detect any cytokines at all you will have to stimulate your cells ex-vivo. 5h with PMA/Ionomycin (at 50ng/ml and 500ng/ml) in your regular growth medium and in the presence of BrefeldinA (e.g. GolgiStop from BD). If your isolation procedure is harsh and you need to be gentle with your cells, keep them in the incuabtor for just a bit (30min) to adjust to the medium, then add PMA/Iono, again incubate for ca. 30min and only then add the BrefeldinA. If your cells are tough and cytokine production is stable you can do it all in one go.
Don't worry about having to do this stimulation, you will still only detect cytokines, which the cells would have been able to produce in vivo. 5h are not enough time to get de novo produced cytokines secreted. Only the translation of already available mRNA will be boosted.
However, it all depends a bit on the cytokines you would like to detect and in which cells. T and B can be done easily with the suggested protocol. IFNy probably won't even need 5h to be detectable, but for e.g. IL-10 you need a bit longer.
Hope this helps!
Hi Maria,
in order to detect any cytokines at all you will have to stimulate your cells ex-vivo. 5h with PMA/Ionomycin (at 50ng/ml and 500ng/ml) in your regular growth medium and in the presence of BrefeldinA (e.g. GolgiStop from BD). If your isolation procedure is harsh and you need to be gentle with your cells, keep them in the incuabtor for just a bit (30min) to adjust to the medium, then add PMA/Iono, again incubate for ca. 30min and only then add the BrefeldinA. If your cells are tough and cytokine production is stable you can do it all in one go.
Don't worry about having to do this stimulation, you will still only detect cytokines, which the cells would have been able to produce in vivo. 5h are not enough time to get de novo produced cytokines secreted. Only the translation of already available mRNA will be boosted.
However, it all depends a bit on the cytokines you would like to detect and in which cells. T and B can be done easily with the suggested protocol. IFNy probably won't even need 5h to be detectable, but for e.g. IL-10 you need a bit longer.
Hope this helps!
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