Live dead fixable blue kit

Flow cytometry analysis was performed using a LSR II and the LSRFortessa X-20 (BD Biosciences) analysers. For analysis, single cell suspensions resuspended in cIMDM. The cells were stained with antibodies in PBS containing 2% FBS for 25 min at 4°C, washed and fixed in 2–4% paraformaldehyde for FACS analysis. For all analysis, viability of the cells was determined using the LIVE/DEAD Fixable Blue kit (Life Technologies). Antibodies used were purchased with eBioscience, Biolegend or BD Pharmingen. They include: CD3 (17A2), CD4 (RM4-5, GK1.5), CD11b (M1/70), CD11c (N418), CD19 (6D5, eBio1D3), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD49b (DX5), F4/80 (BM8), I-A/I-E (M5/114.15.2), KLRG1 (2F1), Ly6G (1A8), NK1.1 (PK136), Sca1 (E13 161.7), SiglecF (E50-2440), TCR β chain (H57-597), TCRγδ (GL3), Ter119 (TER-119), Thy1.2 (53 2.1). Lineage panel for the ILC2 staining was defined as CD4⁺/ CD8⁺/ CD3⁺/ CD19⁺/ TCRβ⁺/ TCRγδ⁺/ NK1.1⁺/ CD49b⁺/ CD11c⁺/ CD11b⁺/ Gr-1⁺/ Ter119⁺. Staining was performed in the presence of FcR Blocking Reagent (Miltenyi Biotec).

Cell suspensions were stained for 25 min with antibodies in PBS with 1% FCS. For flow cytometry analysis, cells were analysed using a BD LSRFortessa™ X-20 (BD Biosciences) or BD LSRII (BD Biosciences) and data were analysed using FlowJo software (Version 10, Treestar Inc). Cells were sometimes fixed in 2–4% paraformaldehyde for FACS analysis. Viability of the cells was determined using the LIVE/DEAD Fixable Blue kit (Life Technologies). Antibodies used include: CD4 (RM4-5; efluor450 (eBioscience)), CD44 (IM7; Percpcy5.5 (eBioscience)), IFNγ (XMG1.2; PE (BD Bioscience)), IL4 (PE, 11B11, eBioscience)), IL5 (APC, TRFK5, BD Bioscience)), IL13 (eFluor660, eBio13A, eBioscience)), IL17a (17B7; PE-Cy7 (eBioscience)), All staining was performed in the presence of FcR Blocking Reagent (Miltenyi Biotec). Intracellular cytokine staining (ICS) was performed following 6 h of re-stimulation with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Promega) and 1 µg/mL ionomycin (Sigma) and BD Golgi Stop and BD Golgi Plug (diluted 1:1000, BD Biosciences). Following surface stain, cells were incubated with eBioscience Fixation/Permeabilization buffer for 25 min followed by 25 min in Permeabilization buffer (eBioscience), and incubation with antibodies in Permeabilization buffer for a further 30 min.