Af 647 conjugated anti foxp3

Negatively isolated CD3⁺ T cells were stained with PE Cy7-conjugated anti-CD4 and PerCP Cy5.5-conjugated anti-CD8 with or without phycoerythrine (PE) or brilliant violet 510 (BV510)-conjugated anti-CD25 (all from BD Biosciences). The cells were permeabilized as per the manufacturer’s instructions and stained with Alexa Fluor (AF)-488 or AF-647 conjugated anti-pS6RP to assess mTORC1 (Cell signaling, Danvers, MA), violet 450 (V450) conjugated anti-pAkt to assess mTORC2 (BD Biosciences), AF-647-conjugated anti-FoxP3 (Biolegend, San Diego, CA), and PE-conjugated anti-GATA-3 (eBioscience). For cytokine detection, FITC-conjugated anti-IFN-γ, allophycocyanin (APC) conjugated anti-IL-4, and PE-conjugated anti-IL-17 were used alone or together. For intracellular cytokine staining, cells were pre-incubated with phorbol myristate acetate (PMA, 5 ng/ml) and ionomycin (500 ng/ml) for 6 hours and with brefeldin A for 5 hours (10 µg/ml; all from Sigma-Aldrich). Isotype control antibodies included FITC-conjugated mouse IgG1, PE-conjugated mouse IgG1 kappa, PE conjugated rat IgG2b kappa, BV510 conjugated mouse IgG1, V450 conjugated mouse IgG1, and APC-conjugated mouse IgG1. All isotype control antibodies were obtained from BD Biosciences except for PE conjugated rat IgG2b kappa, which was obtained from eBioscience.