Apc conjugated anti mouse cd45

Lung cell isolation was performed as previously described [9 (link)]. Lung single cell suspensions were stained with APC-conjugated anti-mouse CD45, PE-conjugated anti-mouse CD103, PerCP-Cy5.5-conjugated anti-mouse CD24 (all from eBioscience, San Diego, CA, USA), Alexa Flour 700-conjugated anti mouse I-A/I-E, BV421-conjugated anti-mouse CD11b (both from Biolegend, San Diego, CA, USA), PE-Cy7-conjugated anti-mouse CD11c, Alexa Flour 647-conjugated anti-mouse siglecF (both from BD Pharmingen, San Diego, CA, USA). Activation of NLRP3 inflammasome was detected as active caspase-1 level using FAM-FLICA Caspase-1 Assay Kit (Immunochemistry Technology, Bloomington, MN, USA). All FACS analyses were performed on a Gallios Flow Cytometer (Gallios, Beckman Coulter, Brea, CA, USA). Alveolar macrophages, interstitial macrophages (IMs), CD11b⁺ monocyte–macrophages/dendritic cells (M–M/DCs) cells and CD103⁺ DCs in lung tissues were isolated using the gating strategy reported by Misharin et al. and Kopf et al. [10 (link), 11 (link)].

3 × 10⁶ RBC-lysed WBM cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with APC conjugated anti-mouse CD45 (clone: 30-F11; eBioscience), PE-conjugated anti-mouse CD201 (EPCR; clone: eBio1560; eBioscience), APC-eFluor780 conjugated anti-mouse CD48 (clone: HM48-1; eBioscience) and Brilliant Violet 421 conjugated anti-mouse CD150 (clone: TC15-12F12.2; BioLegend) antibodies. All antibodies were diluted 1:400. Stained cells were fixed in HSC buffer containing 2% paraformaldehyde overnight at 4 °C. Fixed WBM cells were permeabilized using HSC buffer containing 0.025% Triton X-100 followed by Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Pharmingen). Permeablized WBM cells were stained with a FITC conjugated anti-human Ki67 antibody (BD Pharmingen) and then resuspend in HSC buffer containing 7AAD (BD Pharmingen). Data were collected from 1 million single cells by FACSCanto (BD Pharmingen) and analysed by FlowJo (Tree Star, Inc.) without knowledge of the genotype or treatment by a single observer (C.L.L.).