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Human peripheral blood mononuclear cells (PBMCs), either left unstimulated (left images) or stimulated with 1 µg/mL ionomycin and 20 ng/mL PMA for 2 hours and for additional 4 hours with1 µg/mL brefeldin A at 37 °C, were fixed, permeabilized, and stained with Anti-IFN-γ antibodies as well as with CD69 antibodies. Flow cytometry was performed using the MACSQuant®
Analyzer. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.