Comprehensive Flow Cytometry Analysis of Tissue Cells

Flow cytometry was performed as previously described^{80 (link)}. In short, tissues were cut into 1 mm pieces and digested in collagenase/dispase (Roche) and DNase I (Roche) in Ca2⁺- and Mg2⁺-free Hanks medium plus 5% FCS for 30 min at 37 °C with gentle shaking. Samples were then vortexed for 15 s, filtered and washed in PBS plus 5% FCS. Single cell suspensions were blocked with FC block (Invitrogen) for 20 min at 4 °C, before staining with fluorophore-conjugated primary antibodies (Supplementary Table 3) for 20 min at 4 °C in the dark. Cells were washed twice and re-suspended in PBS supplemented with 5% FCS prior to analysis with either an Aria III cell sorter or BD FACS Canto.
Isotype antibodies (Supplementary Table 3) and fluorescent-minus-one (FMO) controls were used to estimate background fluorescence in combination with either compensation beads and/or unstained controls. Dead cells were detected and excluded from analysis using Sytox Blue or Fixable Viability Dye, eF506. Tuft cells were identified as EpCAM⁺CD45^-/lowCD24⁺SiglecF⁺ (Supplementary Fig. 8a). Inflammatory ILC2s were identified as ST2⁻KLRG1⁺CD90.2⁺Lineage⁻(CD11b⁻CD11c⁻CD19⁻Ly-6G⁻NK1.1⁻CD3^-)CD45⁺. Natural ILCs were identified as ST2⁺KLRG1⁺CD90.2⁺Lineage^-CD45⁺(Supplementary Fig. 8b). All experiments were analyzed with FlowJo software (Version 10).