Isolation and Characterization of Wound-Associated Immune Cells

Spleen and bone marrow were prepared as described previously (Yu et al., 2013 (link)). The peripheral blood monocytes (PBMCs) were isolated using Histoplaque (Sigma). Wound sites were minced and digested with 1.0 mg/ml collagenase (Sigma) to generate a single cell suspension and purified using a Percoll gradient (Sigma). These dissociated single cells were stained with fluorochrome-conjugated antibodies specific for mouse CD11b, Ly6G, CD45 (eBioscience) and CCR2 (R&D). For COL1A1 intracellular staining, cells were harvested, stained with surface markers, fixed with a fixation solution (eBioscience) for 15 min, permeabilized with ice-cold pure methanol for 30 min and incubated with biotin-conjugated mouse COL1A1 antibody (Rockland) and fluorochrome-conjugated streptavidin (eBioscience). Flow cytometry was performed using FACS Aria II (BD) and data analyzed with Flowjo.