Ghost dyetm red 780

Single cell suspensions were blocked with ice-cold 1 × HBSS/5 mM EDTA/1X DNase1/2% goat serum for at least 20 min on ice as previously described (Debuque and Godwin, 2015 (link)). Primary antibodies were then added at final dilution and incubated for at least 1 h on ice shielded from light. Primary antibodies were washed, and if necessary, cells were then incubated with their corresponding secondary antibody for 30 min on ice shielded from light. Cells were then washed and transferred into FACS tubes for flow cytometry analysis or FACS isolation. Cell viability for flow cytometry experiments was assessed with Ghost Dye^TM Red 780 (Tonbo Biosciences, San Diego, CA, United States) following manufacturer’s instructions. Cell viability for FACS experiments was assessed using DAPI. FACS was performed using either the BD symphony A6 or BD Influx Cell sorters. Flow cytometry quantification was performed on LSRII Flow Cytometers (BD Biosciences, San Jose, CA, United States). Compensation of fluorescence spectral overlap was used with UltraComp eBeads (eBioscience, San Diego, CA, United States) according to manufacturer’s instructions. FCS 3.0 files generated by flow cytometry were analyzed using FlowJo Software v10. For list of antibodies used in these experiments and their working dilutions, see Supplementary Table 1.

Single cell suspensions were isolated from mouse lungs by collagenase digestion following recently published protocols (Reyfman et al., 2019 (link)). Briefly, mice were anesthetized with an overdose of 0.5% pentobarbital, then the lungs were perfused with 1 mL complete 1,640 medium (Solarbio) with 10% FCS (Hyclone) containing collagenase IV (Solarbio) and Dnase I (Sigma) through the trachea, chopped with scissors, and subsequently incubated for 20 min at 37°C with mild agitation. The resulting lung homogenate was passed through a 40-μm filter and resuspended in ACK buffer (BD) for 15 min on ice. Then, the cells were centrifuged at 400 g for 6 min and incubated with Percp-Cy5.5 anti-mouse CD45 antibody (BD) and Ghost Dye^TM Red 780 (Tonbo) for 15 min on ice. BD FACS ARIA II cell sorter was applied to sort CD45⁺ cells from lung cell suspensions pooled from three mice.