Complete 1 640 medium

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.

Preparation of cell suspensions followed a recently published protocol [16 (link)]. In brief, an overdose of 0.5% pentobarbital was used to anesthetize mice through intraperitoneal injection. Next, collagenase IV (Solarbio) and Dnase I (Sigma) contained in 1 mL complete 1640 medium (Solarbio) with 10% fetal bovine serum (FBS; Hyclone) were infused into the lung through the trachea. Subsequently, the collected lung tissue was chopped with scissors and incubated for 20 min at 37 °C with mild agitation in the 1640 medium with 10% FBS. After filtration through a 40-µm cell strainer (biolegend), the lung homogenate was spun down at 400 g for 6 min and resuspended in ACK buffer (BD) for 15 min on ice to lyse red blood cells.
After washing with PBS, mouse lung cells were stained with fluorochrome-conjugated antibodies using the recommended concentrations according to instructions provided by the manufacturer. Then, the cells were loaded onto FACS Canto II (BD Biosciences, San Jose, CA, USA) and the resulting FACS data were analyzed by FlowJo (version 10.4, TreeStar, Ashland, OR, USA). Table S1 provides the antibodies used in this research.