Ter119 erythroid cells pe clone ter 119

Macrophages were enriched from isolated wound cells by negative selection. Specifically, cells were stained with Ly6G-PE (clone 1A8; BD Biosciences), CD2-PE (clone RM2–5; BD Biosciences), TER119/Erythroid Cells-PE (clone Ter-119; BD Biosciences), and/or Siglec-F (MerTK^−/− experiments; clone E50–2440; BD Biosciences) for ≤1 hour, washed, and incubated with anti-PE magnetic beads (Miltenyi Biotec) for 20 minutes. Cells were washed and eluted through a magnetic column (Miltenyi Biotec) to deplete non-monocyte/macrophage populations. Enriched monocytes/macrophages were assessed for viability by trypan blue exclusion and surface stained with Ly6C-FITC and F4/80-APC as described above. Eosinophils were identified as F4/80^intSSC^hi and were excluded during sorting. Ly6C^hiF4/80⁺ and Ly6C^lowF4/80^hi populations were sorted to greater than 90% purity under sterile conditions on a FACSAria (BD Biosciences). Sorted cells were assessed for viability by trypan blue exclusion, resuspended in complete medium (DMEM/5% FCS/Penicillin-Streptomycin) and cultured at 37°C/5% CO₂.

Macrophages from the day 14 wound were enriched through negative selection on a magnetic column (Miltenyi Biotec) using the following depletion cocktail: Ly6G-PE (clone 1A8, BD Biosciences), CD2-PE (clone RM2–5; BD Biosciences), Siglec-F-PE (clone E50–2440; BD Biosciences) and TER119/Erythroid Cells-PE (clone Ter-119; BD Biosciences). Staining and column enrichment were carried out as previously described. Following enrichment, wound macrophages were surface stained with Ly6C-FITC, Ly6G-PE, Siglec-F-PE and F4/80-APC for 30 minutes on ice. Sytox Blue (Invitrogen) was used for dead cell exclusion. Ly6C^hiF4/80⁺Ly6G^–Siglec-F^– and Ly6C^low/intF4/80⁺Ly6G^–Siglec-F^– subsets were sorted to ≥90% purity under sterile conditions using a FACSAria.
RNA was isolated from sorted cells using the RNeasy Micro Kit (Qiagen) and cDNA was synthesized with the High Capacity RNA-to-cDNA Kit (Life Technologies). Gene expression analysis was performed by real-time quantitative PCR on a ViiA 7 system (Life Technologies) using TaqMan Assays (Life Technologies) and TaqMan Gene Expression Master Mix (Life Technologies). TaqMan Assay IDs for individual genes are shown in Table S1. Hprt and 18 s served as endogenous controls. Both reference genes produced similar results after normalization. Results using Hprt are shown here. Analysis was carried out using the 2^–ΔΔCt method.