2.3. Identification of ILC2s in sinonasal tissues and peripheral blood
The identification of ILC2s in sinonasal tissues and blood was performed as described previously10,11. Briefly, sinonasal tissues were cut into fine pieces, and digested for 30 to 45 min at 37°C with Liberase TM (125 μg/mL) and DNase I (200 μg/mL; both from Roche Diagnostics GmbH, Mannheim, Germany). Alternatively, cells were isolated by the mechanical disruption of tissues with the gentleMACS Dissociator and Tumor Dissociation Kit (Miltenyi Biotec Inc., Auburn, CA) according to the procedure recommended by the manufacturer. The cell suspensions were filtered through a 70-μm nylon mesh. The remaining cells were treated with ACK lysing buffer (Lonza Corp., Walkersville, MD) to lyse the red blood cells. Peripheral blood mononuclear cells (PBMCs) were isolated with Histopaque 1083 (Sigma-Aldrich, St. Louis, MO).
The total cell suspensions from sinonasal tissues or PBMCs were stained with the following antibody cocktail: fluorescein isothiocyanate-labeled antibodies to lineage markers (CD3, CD11b, CD11c, CD14, CD16, CD19, CD20, CD56, CD123, TCRαβ, TCRγδ, and FceR1α (Lineage)), phycoerythrin-cyanine 7-labeled antibody to CD45, phycoerythrin-labeled antibody to CD127, and Alexa Fluor®-labeled antibody to CRTH2 (BioLegend, San Diego, CA, or eBioscience, San Diego, CA). ILC2s were identified as Lineage-CD45+ CD127+CRTH2+ cells using FACSAria (BD Biosciences, San Jose, CA). The ILC2 prevalence was calculated as the number of ILC2s divided by the total number of Lineage-CD45+ cells.