Isolation of Lung CD45+ Cells

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.