Fixable viability dye efluor 780 or 450

Single-cell suspensions of thymus and spleen were prepared by slide mechanical grind. To isolate lymphocytes in lung and lamina propria, tissues were cut and washed in plain RPMI-1640, and epithelial cells were removed (5 mM EDTA and 1 mM DTT, lamina propria only), followed by enzymatic digestion (0.16 U/ml Liberase TL; Roche) and centrifugation with 47% Percoll gradient to enrich lymphocytes. Cell were stained in FACS buffer (5% FBS in PBS) containing Fixable Viability Dye eFluor 780 or 450 (eBioscience) with the following antibodies for surface staining: CD4, CD8, CD44, and CD62L (eBioscience). To detect cytokine production, cells were stimulated in a 96-well plate with 50 ng/ml PMA, 0.5 µg/ml ionomycin, and 1 µg/ml Brefeldin A (all from Sigma-Aldrich) solution for 4 hat 37°C before staining. Intracellular staining of Ki67, Foxp3, IL-4, IL-17A, and IFN-γ was performed after fixation and permeabilization according to the manufacturer’s instructions. Data were analyzed with FlowJo software (Tree Star).