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.