Primary peritoneal macrophages were harvested in cold DMEMF12 3 to 4 days after intraperitoneal injec tion of 1. 5 ml of 3 % thioglycollate, plated in the presence of 10 % heat inactivated FCS in 96 well culture plates for 2 h, and non adherent cells washed away. The remaining adhered cells are macrophages since they express F480, galectin merely 3MAC 2, and CR3. Myelin isolation Myelin was isolated from mice brains as previously described. C3bi opsonization was carried out by pre incubating myelin in 50 % fresh mouse or rat serum in DMEMF12 for 40 min at 37 C, followed by washing in serum free media as previously described. Levels of phago cytosis were the same whether mouse or rat sera were used, indicating similar opsonization efficiency by the two.
Additionally, experiments Inhibitors,Modulators,Libraries that were performed in the presence of 10 % HI rat serum, 10 % HI mouse serum, 10 % HI FCS, or 0. 1 % delipidated BSA did not differ, indicating that heat inactivation of the complement system was effective in all sera. We used, therefore, mostly rat serum, which is easier to ob tain in the quantities Inhibitors,Modulators,Libraries required to opsonize myelin, and HI FCS as supplement in the phagocytosis assays. The efficiency of C3bi opsonization was tested in each indi vidual experiment by validating that levels of phagocyt osis of C3bi opsonized myelin are about two fold higher than those of unopsonized myelin. Myelin phagocytosis Microglia or macrophages were plated in 96 well tissue culture plates at a density that minimizes cell cell con tact in the presence of DMEMF12 sup plemented with 10 % heat inactivated rat serum, 10 % HI mouse serum, 10 % HI FCS, or 0.
1 % delipidated BSA, thus in the absence of ex ternally provided complement. Phagocytes were left to rest overnight, washed, and myelin was added for the indicated times. Then unphagocytosed myelin was washed out and levels of phagocytosis quantified. At this time all remaining Inhibitors,Modulators,Libraries myelin has already been internalized phagocytosed. When phagocytosis was assayed in the presence of Syk inhibitors, phagocytes were pre incubated in the presence of either SYK inhibitor or piceatannol, or controlvehicle for 15 min, and phagocytosis assayed thereafter in the con tinued presence of inhibitorvehicle. ELISA Inhibitors,Modulators,Libraries assay to quantify myelin phagocytosis is based on the detection of the myelin specific MBP in lysates of phagocytes.
Since MBP is unique to PNS and CNS myelin, and is not produced by phagocytes, MBP levels detected in cytoplasm are pro portional to levels of phagocytosed myelin. In brief, after non phagocytosed myelin is washed away, phagocytes are lysed, lysates transferred to high protein absorbance plates, Inhibitors,Modulators,Libraries and levels of MBP determined by ELISA using rat anti MBP. SB203580 We determined previously that more than 95 % of the detected MBP arises from phagocytosed internalized myelin. We further verified the validity of this phagocytosis assay by testing the ability to detect inhibition of myelin phagocytosis by cytochalasin D.