Isolation of Wound Macrophages and Human Monocytes

MACS of wound cell isolates was performed as described (70 (link)). Briefly, wound cell isolates were incubated with fluorescein isothiocyanate–labeled (FITC-labeled) anti-mouse anti-CD3, anti-NK1.1, anti-CD19, and anti-Ly6G (BioLegend) monoclonal antibodies. Wound isolates were then washed and incubated with anti-FITC microbeads (Miltenyi Biotec, Inc, catalog 130-049-601) and passed through a MACS column (Miltenyi Biotec, Inc). The resultant eluent was then incubated with anti-mouse anti-CD11b microbeads (Miltenyi Biotec, Inc, catalog 130-049-601). The remaining cell population was analyzed by flow cytometry and found to be 97% macrophages, consistent with previous literature (4 (link), 70 (link)). For human monocyte isolation, peripheral blood was collected and subjected to RBC lysis and Ficoll-Paque separation (GE Healthcare). Cell suspensions were then treated with anti-human CD14 microbeads. Magnetic separation yielded 95% purity by flow cytometry.