Fig. 5

Ex vivo assessment of phagocytosis in MDMs and microglia. Ex vivo phagocytosis assays with cultured MDMs and microglia derived from the post-ischemic brain of asplenic mice that received GFP⁺ splenocytes and bead^580/605 at 6th day after stroke. a Cell count distributions for GFP⁺ brain immune cells from contralateral (Contra) or ipsilateral (Ipsi) hemispheres cultured with bead^580/605 at 4 °C or at 37 °C for 4 h. Cell counts with splenocytes cultured at 37 °C with bead^580/605 were used as a negative control. b Measures of phagocytic activity for cultured brain immune cells derived from the contra- and ipsi-lateral hemispheres. Phagocytic activity was monitored by counts of cells containing bead^580/605 (counts), mean fluorescence intensity (MFI) of bead⁺ cells, and a phagocytic index (counts x MFI). The dotted lines in the graphs indicate phagocytic activity of splenocytes incubated at 37 °C as a control. Statistical significance was determined using two-way ANOVA with a post hoc Bonferroni comparison test (^*** and ^****, respectively, indicate p < 0.001 and 0.0001 for the effect of stroke; ^### and ^####, respectively, indicate p < 0.001 and 0.0001 for the effect of temperature). c Measures of phagocytic activity for CD11b⁺/CD45⁺ mononuclear phagocytes in brain immune cell cultures derived from the post-stroke contra- and ipsi-lateral hemispheres (n = 3/group). Statistical significance was determined using one-way ANOVA with a post hoc Bonferroni comparison test (^** and ^***, respectively, indicate p < 0.01 and 0.001). d Representative immunofluorescence images of brain immune cells cultures in phagocytosis assays with arrows indicating phagocytic MDMs [GFP⁺/bead⁺/Iba1⁺] and arrowheads indicating phagocytic microglia or endogenous MDMs [GFP⁻/bead⁺/Iba1⁺]. Scale bar = 20 µm