Figure 1

The activation state affects microglial morphology. A) Increased expression of molecules that are hallmarks of classical activation (iNOS, IL1β) and alternative activation (MRC1/CD206). Rat primary microglial cells were stimulated for 24 hr with 10 ng/ml lipopolysaccharide (LPS) or 20 ng/ml rat recombinant IL4. Gene expression was monitored by quantitative reverse transcriptase (RT)-PCR and normalized to the housekeeping gene, HPRT1. Values are expressed as mean ± SEM for four replicates using different cell cultures. One-way ANOVA with Tukey’s post-hoc test revealed differences from control microglia: ***P <0.001. B) Fluorescence micrographs show that the microglial morphology changes following stimulation with LPS or IL4. Fixed cells were stained with the microglia marker, tomato lectin (FITC-conjugated; green), and nuclei were visualized with DAPI (blue). Examples of unipolar microglia with a fan-shaped lamellum are indicated by arrows, and a bipolar cell is shown by the arrowhead. (Note the 100% purity of the microglial cultures.) Scale bar, 50 μm. C) Differential interference contrast (DIC) micrographs of microglia. For control, untreated microglia, the arrows indicate the smooth leading edge of the lamellum. For IL4-treated cells, the open arrows show regions of membrane ruffling. Scale bar, 50 μm. D) Higher-magnification deconvolved fluorescent images of microglia. The fluorescent labels show F-actin (stained with phalloidin, green), the actin-binding protein, vinculin (red), and cell nuclei (DAPI, blue). The boxed areas were digitally magnified (4×) and shown as insets. Scale bars, 20 μm (main images), 5 μm (insets).