2.10. Flow Cytometry, Intracellular Cytokine Staining, and Transcription Factor Staining

Single-cell suspensions were surface-stained with fluorescently conjugated Abs against murine PerCP-Cy5.5-conjugated CD4 (RM4-5), BV510-conjugated CD8 (53-6.7), BV510-conjugated CD8 (53-6.7), PE-conjugated CD8 (53-6.7), FITC-conjugated CD44 (IM7), APC-conjugated CD62L (MEL-14), or APC-conjugated CD25 (PC61) (BD Biosciences, NJ, USA).

For intracellular cytokine staining (ICS) of BV421-conjugated IFN-γ (XMG1.2) (BD Biosciences), activated T cells were stimulated with leukocyte activation cocktail (BD Biosciences) containing PMA, ionomycin, and the protein transport inhibitor BD GolgiPlug™ (Brefeldin A) for 4 hours, then were first stained for surface markers, followed by ICS with a Cytofix/Cytoperm kit (BD Biosciences).

For transcription factor (TF) staining of PE-conjugated Foxp3 (MF23) (BD Biosciences), activated T cells were first stained for surface markers, then stained with a TF Fix/Perm kit (BD Biosciences).

Flow cytometry was performed on FACSCelesta and LSRFortessa cytometers (BD Biosciences). Analysis was performed with FlowJo software (vX.0.7).