Anti il 17a percp cy5

For flow cytometric analysis of surface markers, single-cell suspensions of lungs were incubated with a mixture containing anti-FcγRIII/II antibody (Biolegend) as well as mouse, rat, and hamster serum to block nonspecific binding. Cells were then incubated with optimal concentrations of the following specific antibodies against surface molecules: anti-CD90.2-APC-eFluor780, anti-CD127-PE-Cy7 (all from eBioscience), anti-CD44-FITC (Biolegend), anti-CD4-V500, anti-CD62L-APC, and anti-KLRG1-BV711 (all from BD Biosciences). For intracellular cytokine staining, 0.8 × 10⁶ cells were stimulated with plate-bound anti-CD3/anti-CD28 (each 5 µg/ml, BD Bioscience) or H1 (5 μg/ml) for 4.5 h in the presence of GolgiPlug™ (BD Biosciences). Cell were stained with optimal concentrations of anti-CD4-V500 (BD Biosciences), anti-CD44-FITC (Biolegend), and anti-CD90.2-APC-eFluor780 (eBioscience). Afterward cells were fixed and permeabilized with Cytofix/Cytoperm™ (BD Biosciences). Intracellularly accumulated cytokines were stained with anti-IFN-γ-PE (Biolegend) or anti-IFN-γ-V450 (BD Bioscience), respectively, anti-TNF-PE-Cy7 (Biolegend), anti-IL-17A-PerCP-Cy5.5 (eBioscience), and anti-IL-2-APC (BD Biosciences). Data were acquired on a FACSCanto™II (BD Bioscience) or on a LSRII (BD Bioscience) and analyzed with the FCS Express 5 Flow Cytometry software (DeNovo™ Software).

BALF or lung mononuclear cells were stained at 4°C in staining buffer (1X PBS, 2% FCS, 2mM EDTA), in the presence of Fc block (2.4G2; BD Biosciences) and analyzed by flow cytometry.
Cells were incubated with CD1d-PBS57-APC tetramers and/or the specific antibodies listed below. For intracellular staining, cells were further fixed with 4% PFA, washed, and permeabilized with 0.5% saponin (Sigma-Aldrich), and then incubated with the anti-cytokine antibodies. The cells were washed and fluorescence was detected using a LSRFortessa (Becton Dickinson). Data were analyzed using the FlowJo 10.4.1 software (Tree Star). Figure S1 represents the gate strategy used.
Antibodies from BD Biosciences: anti-CD3-FITC (145-2C11), anti-CD45-APC-Cy7 (30F11). Antibodies from BioLegend: anti-CD4-Brilliant Violet 605 (RM4-5), anti-CD69-FITC (H1.2F3), anti-CD8a-Brilliant Violet 785 (53-6.7), anti-TCR Vγ1/Cr4-PE (2.11) [Tonegawa 1986 nomenclature, (21 (link))]. Antibodies from eBioscience: anti-CD44-eFluor450 (IM7), anti-TCRb-AlexaFluor 700 (H57-598), anti-IL-13-PE-eFluor610 (eBio13A), anti-IL-17A-PerCP-Cy5.5 (eBio17B7), anti-IL-4-PE-Cy7 (BVD6-24G2), anti-TCRδ-eFluor450 and -PE-Cy7 (GL3), and Fixable Viability Dye eFluor 506.

Cells were isolated from BAL, lungs, spleen, and draining lymph nodes and analyzed by flow cytometry as previously described^{55 (link)}. Cells were stained with the following antibodies at the concentration of 1:200: CD16/32 (i.e., Fc block, eBioscience), Fixable Viability Dye eFluor 506, anti-Ly6G PE-Cy7 or eFluor 450, anti-CD117 FITC, SiglecF PE, CD11b PE-Texas Red, anti-CD11c APC, anti-MHC II AF700, anti-CD49b PerCP eF710, anti-FcεRIα PECy7, anti-F4/80 APC/Cy7, anti-IL17A PerCP-Cy5.5 (Ebioscience), anti-TCRβ APC-CY7 (Biolegend), anti-CD4 Alexa Fluor 700 or eFluor 450, anti-CD8α PE-Texas Red or PerCP-Cy5.5, anti-TCRδ APC, anti-NK1.1 Allophycocyanin, anti-CD44 V500, anti-CD62L PE-Cy7, anti-CD11b Alexa Fluor 647 (BD Pharmingen), anti-B220 Alexa Fluor 700, PE-PBS57 loaded CD1d tetramer was from the National Institute of Allergy and Infectious Diseases Tetramer Facility. Purified anti-CD3 and CD28 antibodies were from BD Biosciences. In some cases, cells were stimulated with PMA and Ionomycin followed by an analysis of intracellular cytokine as previously described^{56 (link)}. Cells were analyzed using a BD FACS Aria II flow cytometer and analyzed with FlowJo software.