Rorγt b2d

Antibodies targeting the following murine proteins were purchased from BioLegend (San Diego, CA): PD-L1 (clone 10F.9G2), I-A/I-E (M5/114.15.2), CD45 (30-F11), CD19 (6D5), CD11b (M1/70), PD-1 (29F.1A12), TCRβ chain (H57-597), CD8a (53-6.7), PD-L2 (TY25), CD80 (16-10A1), CD4 (GK1.5), Ly6G (1A8), Ly6C (HK1.4), T-bet (4B10), ICOS (C398.4A), OX40 (OX-86), TCRγ/δ (GL3) and CD16/32 (clone 93). Antibodies targeting the following murine proteins were purchased from BD Biosciences (Franklin Lake, NJ): Siglec F (E50-2440), CD24 (M1/69), and CD3ε (145-2C11). Antibodies targeting the following murine proteins were purchased from eBioscience (Asheville, NC): iNOS (CXNFT), FoxP3 (FJK-16s), Ki67 (SolA15), CD11c (N418), Gata3 (TWAJ), and RORγt (B2D). A polyclonal antibody targeting murine arginase was purchased from R&D Systems. Isotype control antibodies were used according to manufacturer’s instructions in all experiments. Antibodies were conjugated to the following fluorophores: Allophycocyanin (APC), Alexa Fluor 700, APC-cyanine 7 (APC-Cy7), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein (PerCP)-Cy5.5, PE-eFluor 610, PE-Cy5, PE-Cy7, Brilliant Violet (BV) 421, and BV 650.

Antibodies against the following molecules were used to identify ILC populations: CD45 (clone 30-F11, Biolegend), GATA-3 (TWAJ, eBioscience) and RORγt (B2D, eBioscience). Lineage marker-expressing cells were excluded using a cocktail of FITC-conjugated antibodies purchased from Biolegend against B220 (RA3-6B2), CD3ε (145-2C11), CD4 (RM4-5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD49b (DX5), FcεRIα (MAR-1), Gr-1 (RB6-8C5), NKp46 (29A1.4), TCRβ (H57-597) and TCRγδ (UC7-13D5). Intracellular staining was performed using the Foxp3/Transcription Factor staining kit (eBioscience). Cultured ILC2 were surface stained with Thy1.2 (53-2.1, BD Biosciences), Sca-1 (D7, Biolegend), c-Kit (2B8, Biolegend) and lineage markers, then sorted on a BD FACSAria II on the basis of Lin⁻ Thy1⁺ Sca-1⁺ and c-Kit^low/−. Dead cells were excluded from all experiments based on staining with eFluor 780 fixable viability dye (eBioscience), non-specific Fcγ receptor interactions were blocked with anti-CD16/32 (clone 93, Biolegend), and analyses were restricted to single cells by FSC-H and FSC-W signals. CountBright beads (ThermoFisher) were used to determine cellularity. Cells were analyzed on an LSRII Fortessa or a FACSCalibur cytometer (BD Biosciences) and data were processed using FlowJo version 10.0.8 (Tree Star Inc.).

Cells were isolated from the indicated tissues. Single-cell suspensions were stained with CD16/32 and with indicated fluorochrome-conjugated antibodies. Live/Dead Fixable Violet Cell Stain Kit (Invitrogen) was used to exclude non-viable cells. Multi-laser, flow cytometry analysis procedures were done at the University of Pennsylvania Flow Cytometry and Cell Sorting Facility using BD LSRII cell analyzers running FACSDiva software (BD Biosciences). Two-laser, flow cytometry analyses were done at the University of Pennsylvania iPS Cell Core using BD Accuri C6 instruments. FlowJo software (v.10 TreeStar) was used for data analysis and graphics rendering. All fluorochrome-conjugated antibodies used are listed as follows (Clone, Company, Catalog Number): CD11b (M1/70, Biolegend, 101255); CD11c (N418, Biolegend, 117318); CD16/32 (93, Biolegend, 101319); CD16/32 (93, eBiosciences, 56D0161D80); CD19 (6D5, Biolegend, 115510); CD3ε (145D2C11, Biolegend, 100304); CD4 (GK1.5, Biolegend, 100406); CD45 (30-F11, Biolegend, 103121 or 103151), CD8a (53D6.7, Biolegend, 100725); Foxp3 (FJK-16s, eBiosciences, 50-5773-82); Ly-6G (1A8, Biolegend, 127624); Live/Dead (N/A, Thermofisher, LD34966); NK1.1 (PK136, Biolegend, 108745); RORγt (B2D, eBiosciences, 12-6981-82); Siglec-F (E50D2440, BD, 562757); TCRγδ (UC7-13D5, Biolegend, 107504)