Temporal dynamics and ontogeny of murine FOLR2 ⁺ choroidal macrophages. (A) Representative flow cytometry plots of choroidal macrophages from different aged wild-type mice. (B) Flow cytometry frequencies of FOLR2⁺ choroidal macrophages relative to all immune cells across different aged wild-type mice. n = 8 eyes for 0.5 and 1 mo; n = 9 eyes for 3 mo; n = 10 eyes for 6, 12, 18, and 24 mo. One-way ANOVA with Holm correction. Statistical comparisons were made to the 0.5-mo time point. (C) Log transformed MFI of MHCII expression in FOLR2⁺ macrophages from flow cytometry across different aged wild-type mice. n = 8 eyes for 0.5 and 1 mo; n = 9 eyes for 3 mo; n = 10 eyes for 6, 12, 18, and 24 mo. One-way ANOVA with Holm correction. Statistical comparisons were made to the 0.5-mo time point. (D) Flow cytometry frequencies of MHCII⁺FOLR2⁻, MHCII⁺FOLR2⁺, and MHCII⁻FOLR2⁺ choroidal macrophages relative to all macrophages across different aged wild-type mice. n = 8 eyes for 0.5 and 1 mo; n = 9 eyes for 3 mo; n = 10 eyes for 6, 12, 18, and 24 mo. Two-way ANOVA with Holm correction. Intragroup statistical comparisons were made to the 0.5-mo time point. (E) Whole mount immunofluorescence of choroid/sclera from 0.5 mo MS4A3^TdTomato fate-reporter mouse. Top image scale bar = 500 μm. Bottom image scale bar = 50 μm. (F) Concatenated flow cytometry of mouse choroids from 1, 3, and 6 mo MS4A3^TdTomato fate-reporter mice. (G) Frequencies of total MS4A3^TdTomato-positive choroidal macrophages (MФ) relative to all macrophages. n = 6 eyes per time point. One-way ANOVA with Holm correction. Statistical comparisons were made to the 1-mo time point. (H) Frequencies of MS4A3^TdTomato-positive choroidal macrophages in FOLR2⁺ and FOLR2⁻ macrophage populations. n = 6 eyes per time point. Two-way ANOVA with Holm–Sidak correction. Intragroup statistical comparisons were made to the 1-mo time point. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. MFI; mean fluorescent intensity.