Biotin tcrγδ

Lung single cell suspensions were prepared using a standardized protocol as previously described^{38 (link)}. Cells were stained with the following antibodies from BD Biosciences and eBioscience: eFluor450-CD3 (17A2), PerCP-CD4 (RM4-5), FITC-CD8a (53-6.7), Biotin-TCRγδ (GL3), PE-Streptavidin, APC-IL-17A (eBio17B7), APC-IFNγ (XMG1.2), FITC-CD69 (H1.2F3), PerCP-Cy5.5-Gr1 (RB6-8C5), and PECy7-panNK (DX5). For intracellular staining, cells were stimulated for 5 h with 5 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich; St. Louis, MO, USA) in the presence of a protein-transport inhibitor (GolgiPlug, BD Biosciences; San Jose, CA, USA). Following stimulation, cells were collected, stained with a fixable viability dye (eBioscience), fixed, permeabilized (Fixation and Permeabilization Buffer; eBioscience), then intracellularly stained. Samples were read using a FACSCanto II (BD Biosciences) flow cytometer and analyzed with FlowJo software (version 7.6.5 for Windows; Tree Star; Ashland, OR, USA). To simultaneously compare both quantity (% of cells) and quality (intensity of staining) of IL-17-producing populations, integrated median fluorescent intensity (iMFI) was calculated by multiplying percent of x cell type (i.e., CD4, CD8, γδ, Gr1, NK) of total IL-17A⁺ cells multiplied by the IL-17A MFI of x cell type.