Adult Wistar male rats were randomly assigned into four experimental groups, sham operated, SCI induced, C225 treated, and AG1478 treated. Traumatic SCI was induced by the weight drop technique, as described previously. Briefly, rats were anesthetized with intraperitoneal ketamine and xylazine injections. A T11 SB203580 order spinal lamin ectomy was made to expose spinal cord, and a moderate intensity weight drop was performed by MASCIS Impactor II. Rats in the sham operated group underwent similar proce dures as the SCI induced Inhibitors,Modulators,Libraries group, expect for the weight drop step, rats in both groups were treated with saline through pumps by the following method. Immediately after SCI induction, a subcutaneous osmotic pump was placed closely Inhibitors,Modulators,Libraries to the injury site for intrathecal reagent infusion.
Before plantation, the pumps had been filled with 200 ul saline, C225 or AG1478, connected to a 1. 5 mm long PE 10 tube, then preincubated overnight at 37 C. On day 7 after infusion, the pump was removed and the wound was closed with sutures. Cell culture Highly purified primary microglia cultures Inhibitors,Modulators,Libraries were pre pared using modified methods. Briefly, spinal cords of newborn Wistar pups were dissociated, and cells were carried in mixed cultures for two days. Medium was then refreshed with high glucose DMEM containing 20% fetal bovine serum. Ten days later, microglia cells were isolated by an orbital shaker. Half an hour Inhibitors,Modulators,Libraries after cell implantation, the medium was refreshed for further purification. After identification with CD11b antibody, cultures more than 97% pure were used for experiments.
Since a limited number of primary culture cells are available, BV2 cells were used as succedaneum for western blot analysis. BV2 is an immortalized microglia cell line that is reported to share many characteristics with primary microglia. The cells were cultured in fresh high glucose DMEM supplemented with 10% Inhibitors,Modulators,Libraries fetal bovine serum at a density not exceeding 5 �� 105 cells cm2. Western blot analysis After sacrifice under deep anesthesia by transcardial sa line infusion, experimental rat spinal cord tissue was quickly removed and homogenized by sonication in RIPA lysis buffer. Similarly, cultured cells were lysed by RIPA, scraped off and collected for protein extraction. Lysates were centrifuged at 12,000 x g at 4 C for 10 min and supernatants were collected for the protein concentra tion assay, performed using a BCA kit.
Samples containing 60 ug total protein were loaded on SDS PAGE, and then transferred to nitrocellulose mem branes. After blocking nonspecific binding, blots were incubated with primary antibody overnight at 4 C, fol lowed by conjugation with horseradish peroxidase conjugated immunoglobulin G. Finally, blots were visualized with enhanced chemiluminescence Ponatinib TNKS2 kits, and resulting digital images were analyzed by Image J to obtain the optical densities of signals.