Identification and Quantification of ILC2 in BALF

Cells were analyzed by flow cytometry following staining with live/dead aqua dyes (Live/Dead fixable aqua dead cell stain kit, Invitrogen, 1/1000) and antibodies specific for the following markers: anti-TCRβ (clone H57-597, 5.0 μg/ml) from BioLegend; anti-CD45R/B220 (clone RA3-6B2, 1.0 μg/ml), anti-CD2 (clone RM2-5, 0.8 μg/ml), anti-CD25 (clone PC61, 1.0 μg/ml), anti-CD90.2 (clone 53-2.1, 2.5 μg/ml), and anti-CD11b (clone M1/70, 0.3 μg/ml) antibodies from BD Pharmingen; anti-CD45.2 (clone 104, 0.5 μg/ml) from eBioscience. ILC2 were gated as CD45⁺, CD2⁻ TCRb⁻ B220⁻ CD11b⁻, CD90⁺ and CD25⁺ cells in a similar manner to that described by Roediger et al.^{10 (link)}. Bronchoalveolar lavage fluid (BALF) cells were additionally labeled using anti-Siglec F (clone E50-2440, 2.0 μg/ml), from BD Pharmingen; anti-Gr-1 (clone RB6-8C5, 0.4 μg/ml), anti-CD8 (clone 53-6.7, 5.0 μg/ml), anti-CD4 (clone GK1.5, 1.0 μg/ml), anti-CD3 (clone 2C11, 1.0 μg/ml) from eBioscience; and anti-CD11c (clone N418, 2.5 μg/ml) from BioLegend.