Flow Cytometric Analysis of Lung T-cell Subsets

Cells used for in vitro or in vivo cytokine analysis were stimulated with PMA (20ng/mL; Sigma Aldrich), Ionomycin (1μg/mL; Sigma Aldrich), and Brefeldin A (2μg/mL; Sigma Aldrich) for 4 hours prior to flow staining (W. Lu et al., 2015 (link)). For intracellular staining, cells were fixed and permeabilized using the Mouse FOXP3 Buffer Set (BD) per the manufacturer’s protocol. The fluorophore-conjugated antibodies used in this study were as follows: Live/Dead Fix Blue (Invitrogen), CD3 PerCPCy5.5 (Biolegend), TCRb PE/Cy7 (Biolegend), CD4 PB (Biolegend), CD4 AF700 (Biolegend), CD8 BV650 (Biolegend), CD25 BV421 (Biolegend), FOXP3 AF488 (Biolegend), ROR gamma T PE (Invitrogen), TCF1 AF647 (Cell Signaling Technologies), TCF1 PE (Biolegend), IFNγ AF647 (Biolegend), IL17A FITC (Biolegend), IL17A PE (ebioscience). Representative flow cytometric gating and quantification strategy for detection of lung Th1/Th17 and Tc1/Tc17 cell populations is shown in Supplementary Figure 3. Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).