I understand that to be able to measure intracellular cytokines there is a need to restimulate with PMA and Ionomycin but I don't understand how the results will be reliable after that. PMA and Ionomycin will be able to activate general different pathways and make the cell go crazy and secret different types of cytokines that may not even be relevant to your essay. If you are staining to phenotype your cells or to detect the effect of certain treatment, wouldn't this be affected by the restimulation especially if you are using a TCR targeted activation with CD3/CD28 beads?

Most protocols and vendors recommend re-stimulation and I need to use it too but I am looking for a scientifically justified reason for that!

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