Foxp3 fixation permeabilization buffer kit

Single cell suspensions were prepared from spleen and draining lymph nodes of mice. For transcription factor analysis, 1 × 10⁶ cells were stained for the surface molecules CD4 and γδ TCR on ice for 30 min and washed with ice-cold PBS. Cells were subjected to fixation, and permeabilization with the Foxp3 fixation/permeabilization buffer kit (Biolegend), and intracellular staining for Foxp3, T-bet, RORγt, and Eomes was performed as per the manufacturer's instructions. For intracellular cytokine analysis, cells (6 × 10⁶ cells/well) were stimulated with phorbol myristate acetate (PMA; 50 ng/ml) and ionomycin (850 ng/ml) in the presence of brefeldin-A (5 μg/ml) and monensin (2 μM) in 500 μl/well complete RPMI 1,640 medium in 24-well plates at 37°C in a humidified 5% CO₂ incubator for 6 hours. Cells were collected, washed with PBS, and stained for the surface molecules CD4 and γδ TCR on ice for 30 min, and washed with ice-cold PBS. Cells were subjected to fixation and permeabilization using the Foxp3 fixation/permeabilization buffer kit (Biolegend) and intracellular staining for IL-17A, Foxp3, Eomes, and IFN-γ was performed as per the manufacturer's instructions. Cells were acquired on FACS Canto II (BD Bioscience), and data were analyzed using the FlowJo software.