Intracellular Cytokine Staining for T Cells

For determining T cell-derived cytokine levels, intracellular cytokine staining was performed as described (56 (link)). Briefly, single-cell suspensions from the lungs were stimulated with 2 μg of HA for 5 hr at 37°C. Cells were then incubated with GolgiStop (BD Biosciences) for an additional 5 hr. After wash, cells were incubated with Fc block and then stained with fluorescently-conjugated Abs for T cell surface markers, CD3 PE (145-2C11, BD Biosciences), CD4 APC-Cy7 (RM4-5, BD Biosciences), and CD8 APC-Cy7 (53-6.7, BD Biosciences). Cells were fixed and permeabilized by incubating with BD CytoFix/Perm. After FACS buffer wash, cells were stained for cytokines IFN-γ APC (XMG1.2, BD Biosciences) and TNF-α Alexa Fluor 488 (MP6-XT22, BD Biosciences). All block, incubation, and permeabilization steps were performed for 20 min at 4°C. After wash, cells were suspended in 2% paraformaldehyde and analyzed by flow cytometry as described above.