The lower well contained only medium. After 1 hr, microglia were incubated for 24 hr with ei ther 10 ng ml LPS selleck chemical or 20 ng ml IL4. For the chemotaxis assay, 300 uM ATP was added to the lower well 1 hr after the addition of LPS or IL4. The cell bearing filters were fixed in 4% paraformaldehyde for 10 min, rinsed with PBS, and the microglial cells remaining on the upper side of each filter were removed with a Q tip. The filters were then stained with 0. 3% crystal violet for 1 min, and again rinsed with PBS. The number of cells that had migrated to the underside was counted at 20�� mag nification using an Olympus CK2 inverted microscope. Fibronectin substrate degradation A standard assay for degradation of ECM employs fluorescent labeled substrate on glass coverslips.

ECM degradation is then mon itored as loss of the substrate fluorescence. Inhibitors,Modulators,Libraries We coated coverslips with HiLyte Fluor 488 labeled fibronectin in PBS. After 2 to 3 hr at 37 C, the fibronectin solution was aspirated off, microglia were added and allowed to settle for 1 hr. Standard medium was added, followed 1 hr later by LPS or IL4. After a 24 hr incubation, the cells were fixed and visualized using an Axioplan 2 widefield epifluorescence microscope equipped with an Axiocam HRm digital camera. Invasion analysis Microglial invasion was examined using BioCoat Matrigel Invasion Chambers. These are similar to Transwell chambers, except that the Inhibitors,Modulators,Libraries 8 um diameter holes in the upper filter are coated with Matrigel, which is a base ment membrane type of ECM secreted by mouse sar coma cells. Microglia in fresh standard medium were added to the upper well.

After 1 hr incubation, 10 ng ml LPS or 20 ng ml IL4 was added to the experimental wells. When used to stimulate chemotaxis, 300 uM ATP was added to the lower chamber of wells after another 1 hr incubation. All chambers were then incubated for 24 hr. Statistical analysis Inhibitors,Modulators,Libraries Quantitative data are presented as mean SEM, and an alyzed with either one way analysis of variance, followed by Tukeys post hoc test or two way ANOVA with Bonferroni correction. GraphPad Prism ver 5. 01 was used. Inhibitors,Modulators,Libraries Results are considered significant if P 0. 05. Results The microglial activation state affects their morphology In order to analyze functional outcomes of different acti vation stimuli, we have established culturing methods that maintain a relatively resting state with low produc tion of cytokines and reactive oxygen and nitrogen species. Here, untreated primary rat microglia had very low expression of the three activation markers, inducible nitric oxide synthase, IL1B, mannose receptor 1. LPS selectively induced the Inhibitors,Modulators,Libraries classical activation Erlotinib EGFR markers, iNOS and IL1B, while IL4 selectively induced the alternative activation marker, MRC1, as before.