Tissue Preparation and Flow Cytometry

The cell suspension obtained in the previous section was preincubated with Mouse BD Block purified antimouse CD16/CD32 mAb (394,656; clone: 2.4G2; 1/100; BD Biosciences) for 10 min at 22°C. The following antibodies were used for the gating of the innate lymphoid cells. Cell suspensions were incubated with a mixture of Biotin-CD3e (100,304; clone: 145-2C11; 1/200; eBioscience, Inc.), Biotin-CD45R/B220 (103,204; clone: RA3–6B2; 1/200; eBioscience, Inc.), Biotin-Gr-1 (108,404; clone: RB6-8C5; 1/200; eBioscience), Biotin-CD11c (117,304; clone: N418; 1/200; eBioscience, Inc.), Biotin-CD11b (101,204; clone: M1/70; 1/200; eBioscience, Inc.), Biotin-Ter119 (116,204; clone: TER-119; 1/200; eBioscience, Inc.), Biotin-FceRIa (134,304; clone: MAR-1; 1/200; eBioscience, Inc.), Brilliant Violet 510-Streptavidin (405,233; 1/500; eBioscience, Inc.), PE-Cy7-CD127 (135,014; clone: A7R34; 1/100; eBioscience, Inc.), Pacific Blue-CD45 (103,116; clone: 30-F11; 1/100; eBioscience, Inc.), and Fixable Viability Dye eFluor 780 (1/400; eBioscience, Inc.) for 20 min at 4°C. The cell suspension was washed twice with 2% FBS/PBS and fixed with fixation buffer (420,801; BioLegend, Inc.) for 30 min. After washing with 2% FBS/PBS, the cell suspension was incubated with the mixture of PE-GATA-3 (clone TWAJ, 1/50; eBioscience, Inc.), APC-RORγ (clone AFKJS-9, 1/50, eBioscience, Inc.), and FITC-T-bet (clone 4B10, 1/50; BioLegend, Inc.)^60,61 (Figure S1). We used the following antibodies for gating of M1 and M2 macrophages: FITC-CD206 ({"type":"entrez-nucleotide","attrs":{"text":"MA516870","term_id":"1543283220","term_text":"MA516870"}}MA516870; clone: MR5D3, 1/50, eBioscience, Inc.), PE-F4/80 (12,480,182; clone: BM8, 1/50, eBioscience, Inc.), APC- CD45.2 (17,045,482; clone: 104, 1/50; eBioscience, Inc.), PE-Cy7-CD11c (25,011,482; clone: N418, 1/50, eBioscience, Inc.), and APC-Cy7-CD11b (47,011,282; clone: M1/70, 1/50; eBioscience, Inc.)⁶² (Figure S2). We analyzed the stained cells using flow cytometry Canto II, and the data were analyzed using FlowJo (version 10; TreeStar, Inc.) (n=10).