Fig. 5.

Culture media impacts differentiated MDM morphology and cell cycle state. (A) Representative bright-field images of MDMs differentiated in all 5 media at baseline on days in vitro 6 or 7. Blue arrows denote spindled/elongated morphology and red chevrons highlight rounded morphology. High content quantification of morphological features at baseline includes (B) macrophage area (n = 10–18), (C) macrophage roundness or P2A (n = 10–16), and (D) macrophage elongation or LWR (n = 11 to 17). (E) Representative images of high content imaging to assess proliferation marker, Ki-67, expression on macrophages cultured in each media condition. (F) High content quantification of Ki-67+ cells (n = 8–12). Bright-field images captured with a Nikon NIS Elements microscope, 20 × objective at room temperature. Immunofluorescence images captured with CX7 Automated High Content Imager, 20×. Image analysis was performed using HCS Studio 2.0 using the Morphology and Target Activation Bio-applications. Parameters for image analysis are shown in Table 2. Data from all wells were pooled for analysis. Statistical analyses used Kruskal-Wallis test with Dunn's multiple comparisons for post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001.