Flow Cytometric Analysis of BALF

Differential cell counts in BALF were determined by flow cytometry as described [26 (link), 27 (link)]. Briefly, BALF cells were resuspended in FACS buffer (5% BSA, 0.35 mM EDTA, 0.01% NaN3). Cell staining was performed according to manufacturer’s recommendations using fixable viability dye eFluor 780, rat anti mouse-CD16/CD32 (clone 93), rat anti mouse-CD45 PE-eFluor610 (30-F11), rat anti-mouse CD11b PE-Cy7 (clone M1/70), rat anti-mouse Siglec-F Alexa Fluor 647 (clone E50-2440) (all from BD Biosciences, San Jose, CA); anti-mouse CD64 PerCP-Cy5-5 (clone X54-5/7.1) and rat anti-mouse Ly-6G FITC (clone 1A8) (both from Biolegend, San Diego, CA). Flow cytometry analysis was performed using a FACSCANTO II (Becton Dickinson, Franklin Lakes, NJ) and data were analyzed using FlowJo software (Becton Dickinson). The gating strategy that was used to analyze the different populations is shown in supplementary Figure S1. Fibrotic macrophage population was defined as CD45 + CD64 + SigecF^lowCD11b^hi; classical alveolar macrophages were defined as CD45 + CD64 + SiglecF^hiCD11b^low.