Figure 3. Characterization of macrophage recruitment during ADRB3 stimulation. (A) Single-cell images of MGL1+F4/80+ M2 macrophages obtained from ImageStream. Cytoplasmic lipid droplets are visualized in bright field images and LipidTox staining. Nuclei were counterstained with DAPI. (B–E) Flow cytometric characterization of F4/80+ macrophages in gWAT from control mice and mice treated with CL for 3 d. Mice were injected with EdU 2 h before sacrifice (n = 4–6 per condition, mean ± S.E.M). (B) Representative histograms and quantification of F4/80^hi and F4/80^lo macrophages in SVC from gWAT. Contents of F4/80^hi macrophages significantly increased after 3 d of CL treatment (***P < 0.001). (C) Analysis of EdU incorporation in F4/80^hi and F4/80^lo macrophages, showing a significant increase in the mitotic index of F4/80^hi macrophages (***P < 0.001). (D) Analysis of CD44 expression in F4/80^hi and F4/80^lo macrophages, showing upregulation of CD44 expression in F4/80^hi macrophages after 3 d of CL treatment (*P < 0.05). (E) Analysis of lipid content and CD44 expression in F4/80+ macrophages. MFI of lipid staining and CD44 expression showed a strong positive correlation.