Single-cell suspension for cell culture, ImageStream flow cytometry, flow cytometry, and scRNA-seq.

Whole lungs were removed and put into complete DMEM with 1 mg/mL collagenase type II (Thermo Fisher Scientific, NC9693955). Complete DMEM consisted of 10% FBS (Invitrogen), penicillin/streptomycin (Invitrogen, 15-140-122), vitamins (Invitrogen, 11120052), Glutamax (Invitrogen, 10566016), nonessential amino acids (Invitrogen, 11140050), and sodium pyruvate (Invitrogen, 11-360-070). Lungs were incubated at 37°C for 30 minutes and then mashed through a 100-μm cell strainer (Thermo Fisher Scientific, 08-771-19). ACK Lysing Buffer (Thermo Fisher Scientific, A1049201) was added to the single-cell suspension and this mixture was spun down at 135g for 5 minutes. The ACK was washed out with 50 mL of isolation buffer, which consisted of 1 mM EDTA and 2.5% FBS in PBS.

For Mk isolation, a negative-selection protocol was used that included biotinylated anti-CD11b (BioLegend, NC0200884), -B220 (BioLegend, NC0200885), -CD3ε (BioLegend, 100304), and -CD146 (BioLegend, 134716) antibodies and incubating in a 5 mL polystyrene tube (Laboratory Product Sales, L285601) with 50 μL/mL of rat serum. All antibodies were at 5 μg/mL final concentration and streptavidin beads (STEMCELL Technologies, 19860) at 75 μL/mL of sample (under 1 × 10⁹ cells) concentration added. Each sample was then added to a magnet and incubated for 3–5 minutes and non-bound cells transferred to a 15 mL tube with prewarmed complete DMEM.

Cells from tibias and femurs were isolated by flushing the BM with isolation buffer using a 20-gauge needle (BD Biosciences, 14-826D). If Mks were used for experimentation, the same procedure that was used for the lungs was used for the BM. For BMDCs we followed the protocol provided by Abcam (https://www.abcam.com/protocols/bmdc-isolation-protocol).