Cell surface marker and intracellular SPC expression was analysed by flow cytometry as described (Kamata et al., 2015, 2020). Primary antibodies used were as follows: anti-mouse CD11b (clone M1/70, Tonbo Biosciences; 1:300), anti-Gr1 (clone RB6-8C5, SouthernBiotech; 1:500), anti-mouse CD11c (clone N418, BioLegend; 1:200), anti-F4/80 (clone BM8, BioLegend; 1:100), anti-Ly6C (clone HK1.4, BioLegend; 1:200), anti-mouse CD45 (clone 30-F11, BioLegend; 1:600), anti-mouse CD4 (clone GK1.5, BioLegend; 1:100), anti-mouse CD8a (clone 53-6.7, BioLegend; 1:200), anti-mouse B220 (clone RA3-6B2, BioLegend; 1:200), anti-mouse CD31 (clone MEK13.3, BioLegend; 1:200) and anti-Sca1 (clone D7, Miltenyi Biotech; 1:300) antibodies. Stromal cell types quantified using this panel were previously described (Kamata et al., 2020). For SPC intracellular staining, surface-stained lung cells were fixed/permeabilised using a BD Cytofix/Cytoperm™ kit (BD Biosciences), according to the manufacturer's instructions, and frozen at −20°C for 24 h. Then, the frozen cells were thawed in a 37°C water bath and stained with an anti-SPC antibody (FL-197, Santa Cruz Biotechnology; 1:100) in BD Perm/Wash™ buffer (BD Biosciences) at 37°C for 45 min followed by AlexaFluor®488-conjugated anti-rabbit antibody (Thermo Fisher Scientific; 1:2000) staining in BD Perm/Wash™ buffer at room temperature for 20 min.
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