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). TableS1 provides the antibodies used in this research.
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
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