Multicolor Flow Cytometry for Macrophage Polarization

For the macrophage polarization analysis, a multicolour flow cytometry protocol with a sequential gating strategy was developed as previously described [15 (link)]. Cell suspensions were stained with antibodies specific for CCR7 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD86 (BioLegend, Sen Diego, CA, USA), CXCR1 (R&D Systems, Minneapolis, MN, USA), and CCR2 (BioLegend), and the cells were then fixed and permeabilized with Cytofix–Cytoperm solution (BD Biosciences). In the PM-2 K⁺ cell population, cells that were CCR7⁺CD86⁺ were defined as M1-like macrophages, while those that were CCR7⁻CXCR1⁺, CCR7⁻CD86⁺, and CCR7⁻CCR2⁺ were defined as M2a-, M2b-, and M2c-like macrophages, respectively [3 (link), 15 (link)]. The histograms with all the antibodies and gating strategies used in flow cytometry for the macrophage subsets are shown in Additional file 1.