I was setting up a flow cytometry assay for intracellular interferon-g (IFNg), using mouse splenocytes stimulated for 4 hrs with PMA/ionomycin, the last 3 hrs in the presence of Brefeldin A. I was surprised to see a population of positive cells in the non-stimulated cells. The IFNG+ cells were CD8 negative. PMA/ionomycin increased the percentage of IFNg positive cells in both CD8+ and CD8- populations, but I was curious whether the IFNg cells are legitimate; they are about 2% of all cells.
i think that interferon gamma is a natural chemokine or cytokine produced by certain type of T cellsand i guess that this result is normal
PMA/Ionomycin will stimulate all cells with the capacity to produce IFN-g and other cytokines. Therefore, you result is not surprising. How do you define stimulation? I think this is a semantic question. In my wording, the IFN-g producing cells would be among the activated cell Population
Dear Edward, hundreds of immune responses to different foreign and self antigens can take place at the moment. There is no surprising! You should see them on the level of IFNgamma production. I think, you will see them even in germfree mice.
I think that's quite believable! However, in case you are not sure, the obvious source of that signal other than legitimate IFNg is non-specific signal from the excess antibody that wasn't washed out of the cells. ICS is always suspicious in that regard.
Grab some "isotype" (in fact, non-matched isotype would also work) antibody and run it in parallel in unstim cells. If you don't see the positive cells you are good! If you see some, attribute those 2% to "unwashed background" and see if you could improve your blocking/washing steps. Sometimes you just can't!
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