I am studying a chronic condition (diabetes) in human samples and preliminary ex-vivo results show a difference in cytokines secretion. I would like to do ICS to define the subpopulations that contribute to this. Surface staining already shows that there are no differences in cell frequencies, so what I am likely looking for is functional changes in existing populations. My concern therefore is that using PMA/Iono would mask these fine differences by activating everything. Would using BFA only for a few hours/overnight after cell isolation be sufficient to reliably measure ICS? Does anyone know of any resource in support of this idea (or convincingly contrary to it, then I can put my mind to rest)? I cannot find any publication describing such a protocol, and unfortunately I am very limited in accessing samples so I cannot run extensive tests.