After the indicated times in culture, the frequency of cytokine-producing T cells was determined by ICS. Briefly, 0.5–1.0x106 cells were stimulated in media containing PMA and ionomycin or plate bound anti-CD3 as previously described (25 (link)). After 2–3 hours monensin (2μM) was added to stimulated cells and 3 hours later, cells were stained with a fixable viability dye (eBioscience) and fixed with 4% formaldehyde at room temperature for 10 minutes. After fixation, cells were permeabilized with permeabilization buffer (eBioscience) and stained for intracellular cytokines (IL-4: 11B11 [BioLegend], IL-9: RM9A4 [BioLegend], IL-10: JES5-16E3 [BioLegend], and IL-17A: eBio17B7 [eBioscience]) in the same buffer.
Staining for intracellular STATs was done after fixation with 4% formaldyhyde, as above, and permeabilization with cold 100% methanol. Cells were subsequently washed with PBS and stained with flourochrome-labeled antibodies to total STAT3, pSTAT3(Y705) and pSTAT5(Y694) (all from BD Biosciences).
Staining for intracellular STATs was done after fixation with 4% formaldyhyde, as above, and permeabilization with cold 100% methanol. Cells were subsequently washed with PBS and stained with flourochrome-labeled antibodies to total STAT3, pSTAT3(Y705) and pSTAT5(Y694) (all from BD Biosciences).