FACS was performed on an LSRII flow cytometer on digested lung tissue suspensions prepared according to a method already in use in our lab (51). The mice of interest were euthanized and the lungs were flushed with 1× PBS as described above. Lungs were harvested, chopped into small pieces, and then digested in DMEM with high glucose containing 150 μg/mL collagenase (MilliporeSigma, C5138) and 20 U/mL DNase I (Roche, 04716728) for 1 hour at room temperature. This digestion was halted by addition of 1× PBS. The digested tissue was mechanically dissociated, filtered through a 100-μm cell strainer, and then centrifuged at 495g for 10 minutes at 4°C. The supernatants were dumped, and 10 mL of 1× PBS were added into the pellets for cell counting. After centrifuging again, the pellets were resuspended in FACS buffer (1× PBS containing 2% FBS, 0.01% NaN3, and 1 mM EDTA) at a density of 2 × 107 cells/mL. Next, 2 × 106 cells were taken and incubated with the surface antibodies in FACS buffer containing 10% NGS for 1 hour at 4°C. Antibodies are shown in Supplemental Table 4. The following surface antibodies were used: rat CD45 mAb FITC (1:100, eBioscience, 11-0451-82), rat PE CD45 (1:100, BD Biosciences, 553081), rat CD45 mAb PerCP (1:1000, eBioscience, 45-0451-82), rat CD45 APC (1:100, eBioscience, 17-0451-82), rabbit anti-ADRA1A (1:5000, Abcam, ab22519), rabbit anti-ADRA1B (1:1000, Abcam, ab169523), rabbit alpha-1D adrenoceptor (1:100, Invitrogen, PA5-77286), rat PE CD31 (1:100, BD Biosciences, 553373), rat CD326 (EpCAM) (G8.8) APC (1:200, Invitrogen, 17-5791-82), rat PE anti-CD11b (1:200, BD Biosciences, 557397), Armenian-hamster CD11c (N418) PerCP-cyanine 5.5 (1:800, eBioscience, 45-0114-80), and rat F4/80 (BM8) APC (1.5:100, Invitrogen, 17-4801-82). After washing with FACS buffer, cell pellets were permeabilized in 500 μL of 1× fixation/permeabilization buffer (eBioscience, 005123-43) overnight at 4°C. The next day, cells were washed with 1× permeabilization buffer (eBioscience, 00-8333-56) and then continuously labeled with intracellular antibodies for 1 hour at room temperature. The following intracellular antibodies were used: rat FITC CD206 (MRC1) (1.5:100, BioLegend, 141704), goat anti–type I collagen (1:100, Southern Biotech, 1310-01), and mouse netrin-1-FITC (1:200, Novus Biologicals, NB600-1344F). Where relevant, secondary detection of unconjugated primary antibodies was then performed. Finally, cells were washed with FACS buffer and filtered through a 100-μm cell strainer. Acquisition was performed on an LSRII flow cytometer using FACSDiva acquisition software. FlowJo software (FlowJo, LLC) was used for data analysis. The positive and negative gates were set based on relevant isotype control. Antibodies are listed in Supplemental Table 3.

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