Flow Cytometry

For BAL fluid collected, leukocyte populations were gated as previously described (33). Single cell suspensions of lung cells were stained for ILC2 as Live CD45⁺ Lineage⁻ Thy1-2⁺ CD127⁺ ST2⁺. For cytokine detection cells were stimulated for 4 h with PMA Ionomycin and Brefeldin A (cell stimulation cocktail, Biolegend) or BrefeldinA alone. After 15 min of viability stain with GhostRED (Tonbo, San Diego CA), cell fixation and intracellular cytokine staining was performed according to manufacturer instructions (BD biosciences San Jose CA) using IL-5-BV421, IL-13-APC, IL-17a-PeCy7 (Biologend, San Diego CA). For Transcription factor staining, FOXP3 fixation permeabilization kit was used according to manufacture and stained with GATA3-PECy7, ki67-e450, and IRF4-PE (eBioscience San Diego, CA). For mitochondrial analysis, ILC2 cultured with 20 nM MitoTrackerGreen and 100 nM MitoTrackercmxROS-H₂ or 5 uM mitoSOX (Invitrogen, Carlsbad CA) in PBS with GhostRED dye for 30 min at 37°C 5% CO₂. Cells were acquired on BD Canto II and analyzed with FlowJo software (Treestar Ashland OR).