Figure 4. Serum exposure unlocks latent phagocytic potential in serum-free microglial cultures.

(A) Phagocytosis (3 hr) of pHrodo-labeled myelin debris (red) by primary rat microglia (5 div in TIC) with (middle, bottom) or without (top) exposure to serum (10% FCS, 24 hr). (B) pHrodo signal monitored over two days. Microglia (5 div in TIC) exposed to 10% FCS for 24 hours before addition of myelin (black) cleared all of the provided myelin. Cells only exposed to 10% FCS when myelin was added (red) were initially less phagocytic than cells pre-exposed to serum, but gradually acquired robust phagocytic capacity. Cells unexposed to serum (gray) showed minimal myelin uptake. (C) Serum-free cultured microglia (5 div in TIC) do not phagocytose myelin pre-opsonized with serum (Pre-Ops. Myelin, 4 hr). Pre-exposure of microglia (24 hr) to adolescent rat serum or fetal calf serum facilitates phagocytosis. Minimal pHrodo signal was observed in the absence of myelin or in the presence of the actin-polymerization inhibitor cytochalasin D (CytoD, 10 μM). Phagocytic index was calculated as the ratio of pHrodo+ area to calcein+ area. (D) 8 or 48 hr after removal of FCS (10%, 24 hr exposure), microglia phagocytose myelin (4 hr) as efficiently as cells continuously maintained with serum. (E) Microglia (5 div in TIC) supplemented with serum before adding myelin (10% FCS at t = −16 hr) or with serum and the translational inhibitor cycloheximide (40 μM, FCS and CHX at t = −16 hr) show that serum-evoked changes in phagocytosis requires new protein synthesis. (F) Microglia (0 div in TIC) do not phagocytose myelin immediately after plating, although phagocytosis gradually increases in cells exposed to 10% FCS. Averages are mean ± sem. Scale bars = 10 μm. * P < 0.001 one-way ANOVA with Dunnett’s comparison to no FCS.