Isolation and flow cytometry analysis of mouse lung cells

After measuring PAP, mouse lungs were perfused by right ventricle administration of PBS, dissected, and digested as previously described [28 (link)]. Briefly, left lobes were digested with Liberase TM (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany), disrupted, and filtered using a 40 µm cell strainer (Corning^®, Somerville, NY, USA), followed by centrifugation for 10 min at 300× g. Red blood cells were lysed with ACK lysis buffer, and cells were resuspended in RPMI medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), filtered again, centrifuged, and resuspended in RPMI. Cell counting was performed using a Neubauer chamber after staining with trypan blue to exclude dead cells. For flow cytometry analysis, cells were resuspended in FACS buffer (PBS containing 5% bovine serum albumin and 0.1% sodium azide). Following the blocking of Fc receptors with anti-CD16/CD32 antibodies (BD Biosciences, San Jose, CA, USA), cells were stained with fluorochrome-conjugated antibodies to extracellular CD45, CD4, CD3, TCRβ, TCRδ, and intracellular staining for IL-6, IL-13, IL-17, and IFN-γ, all from BD Biosciences. Data were acquired with a BD FACSCalibur flow cytometer and analyzed using FlowJo™ v10 Software (BD Biosciences, FlowJo, LLC, Ashland, OR, USA) following the gating strategy as indicated in Supplementary Figure S2.