Facscanto 3

The anti-mouse TCRβ (mTCR)- PerCp-Cy5.5, CD8a-eFluor 450, were purchased from eBioscience. CD3-APC-Cy7, CD4- PE/FITC/APC, 4–1BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD (Becton Dickinson Biosciences). CD3-Alexa Fluor 700, CD62L-PE-Cy7, and the live/dead stains- DAPI/PI were purchased from Biolegend.
For FACS sample preparation, cells were harvested and washed with FACS buffer. Cells were resuspended in FACS buffer at a concentration of 1e6–50e6 cells/ml and extracellular fluorescent-conjugated primary antibodies were added and mixed in. After a 20–60 min incubation at 4^oC, which was also protected from light exposure, the samples’ cells were washed and resuspended with FACS buffer.
The flow cytometry assays were performed on FACSCanto I/II (BD Biosciences) and the acquired data were analyzed with FlowJo software (TreeStar). Cells were sorted to: Live/CD3⁺, separated for CD4⁺ or CD8⁺, T_EMRA/ T_EM/T_CM based on sorting out CD62L⁺/CD45RO^- (T naïve cells). Cocultures were sorted for enrichment or into single reactive cells based on 41BB⁺/OX40⁺ (both double and single-positive cells), Live/CD3⁺, separated for CD4⁺ or CD8⁺ by SH800S/MA900 instrument (Sony Biotechnology) or by FACSAria II (BD).

Lung cells were isolated as previously described and cell viability was confirmed by trypan blue exclusion^{39 (link)}. To assess cytokine production by T-cells, approximately 500,000 lung cells were added to a 96-U-well plate in duplicate before being stimulated with PMA (50ng/ml; Sigma) and ionomycin (500ng/ml; Sigma) and incubated at 37C for 4h. After the first hour BrefeldinA (1000X; eBioscience) was added to the culture to block secretion. Following ex vivo stimulation, cells from duplicate wells were pooled and transferred to a 96-V-well plate on ice, centrifuged, and resuspended in PBS containing TruStain fcX (anti-mouse CD16/32; BioLegend). T cells were stained with anti-mouse CD4–fluorescein isothiocyanate (FITC), CD3–AlexaFluor 700, γδ TCR-phycoerythrin(PE)/Cy7 and CD44–Pacific Blue (BioLegend). Cells were labeled with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen by Life Technologies, Carlsbad, CA). Intracellular staining for IL13-phycoeruthrin(PE), IL17A–AlexaFluor 647, and IFNγ–PerCP5.5 was conducted according to the manufacturer’s instructions (eBioscience, San Diego, CA). Acquisition was done on a FACSCanto III (Becton Dickinson, Mountain View, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR).