Spatial and functional analyses of macrophage subsets and analysis of intratumoral cytokines. (A) Gating strategy identifying MHC-II^Hi, Arg1^Hi, and CD206^Hi macrophage subsets used for flow cytometric analysis and FACS sorting. CD206^Hi TAM and non-CD206^Hi populations on the fourth plot were used for co-culture T cell suppression assay. Analysis of subcutaneous CIT9-RFP tumor is shown as a representative data. Representation of at least three independent experiments. (B) Spatial infiltration pattern of macrophage subsets in mixed-population tumors measured by microdissection followed by flow cytometry, presented as % total macrophages (n = 8–9). The experiment was performed once. (C) CD69⁺ CD25⁺–activated T cell count in CD206^Hi or non-CD206^Hi macrophage–T cell co-culture (1:16 and 1:4 ratios) experiment (n = 3–6). Data are a representation of two independent experiments. (D) Protein expression of 43 cytokine and chemokines in CIT6-YFP and CIT9-RFP tumors measured by ELISA (n = 3). Experiment was done once. Statistical significance was determined by one-way ANOVA (B) or two-way ANOVA (C) with Tukey’s correction or by Student’s t test (D). *P < 0.05, **P < 0.01, and ***P < 0.001.