Il 2 pe cf594

For analysis of PD-1, cells were washed in serum-free PBS and stained with a fixable viability dye, eFluor506, CD4-PerCPCy5.5, PD1-PECY7, CD8-APCeFluor780 (eBioscience), incubated for 15 min at room temperature in the dark, and then washed in PBS buffer containing 1% FBS and sodium azide. Cells were stained for Treg cell markers using a FoxP3 staining buffer set (eBioscience) and accompanying protocol. Treg cell markers included CD39-FITC, FoxP3-PE, CD73-PerCPeFluor710, CD25-PECY7, CTLA-4-APC, CD127-APCeFluor780, Ki67-eFluor450 (eBioscience), and CD4-V500 (BD Biosciences). An intracellular staining kit (Fix and Perm kit, Invitrogen) was used to analyze cytokine production after restimulation with PMA/ionomycin. Cells were stained with IL-17A-AlexaFluor488, IL-10-PE, TNF-α-PerCPCy5.5, CD45RA-PECY7, CD8-APCeFluor780, FoxP3-eFluor450 (all eBioSciences), CD45 AlexaFluor700 (BioLegend), IFN-γ-APC, CD3-V500, and IL-2-PE-CF594 (BD Biosciences). Due to PMA/ionomycin-mediated reduction in CD4 expression, CD4⁺ T cells were identified as CD3⁺CD8⁻ T cells for cytokine analysis. Cells were acquired on a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Flowjo LLC).