Apc cy7 conjugated rat anti mouse cd45 antibody

For intracellular EdU cytometry flow, C57BL/6 mice were injected intraperitoneally with EdU (50 mg/kg; Santa Cruz Biotechnology, #sc-284628) at indicated time points. After euthanasia, the whole lung single-cell suspension was prepared as above and fixed by 3.2% paraformaldehyde (PFA) (Electron Microscopy Sciences, #15714-S) for 15 min, washed twice using 3% bovine serum albumin (BSA) (in PBS), and permeabilized using 0.1% Triton X-100 (in PBS) for 15 min. EdU was detected using the Click-iT reaction coupled to an Alexa Fluor 647 azide following the instructions of the manufacturer (Invitrogen, #C10086). For VECad-CreERT2 lineage analysis, lung cell suspensions were prepared as above. Cells were blocked in SB containing 1:50 TruStain FcX (anti-mouse CD16/32) antibody (BioLegend, #101319) for 10 min at 37°C. The cell suspension was stained using APC/Cy7-conjugated rat anti-mouse CD45 antibody (1:200; BioLegend, #101319), Alexa Fluor 488–conjugated rat anti-mouse CD31 (PECAM1) antibody (1:200; BioLegend, MEC13.3, #102513) for 45 min at 4°C. Intracellular flow analyses were performed on BD FACS Canto II flow cytometer (BD Biosciences).

Whole lung single-cell suspensions were prepared as above and then blocked in SB containing 1:50 TruStain FcX (anti-mouse CD16/32) antibody (BioLegend, #101319) for 10 min at 37°C. The cell suspension was stained using allophycocyanin (APC)/Cy7–conjugated rat anti-mouse CD45 antibody (1:200; BioLegend, #101319), Alexa Fluor 488–conjugated rat anti-mouse CD31 [platelet endothelial cell adhesion molecule 1 (PECAM1)] antibody (1:200; BioLegend, MEC13.3, #102513) for 45 min at 4°C. Stained cells and “fluorescence minus one” controls were then resuspended in SB + 1:1000 DNase + 1:1000 Draq7 (BioLegend, #424001) as a live/dead stain. All FACS sorting was performed on a BD FACSAria Fusion Sorter (BD Biosciences).