Connective tissue and fat were MK-2206 dissected away from muscle tissue under a dissecting microscope and muscle was weighed on an analytical balance. Isolation of the myoblastic population was performed as previously described, with the following modifications and details. Diced muscle was minced for 2 minutes using curved-tip scissors and treated with 50 mL of 0.05% trypsin- EDTA diluted in Hank��s saline per mg of tissue. The tissue/trypsin mixture was incubated at 37uC and pipetted thoroughly every 5 minutes for 20 minutes total. The trypsinized tissue mixture was then passed through a 70 mm cell strainer to reduce fibroblast contamination and the strainer was rinsed with an additional 100 mL of F10C per mg of tissue. The flowthrough was then centrifuged at 800 rpm for 5 minutes and plated on gelatin-coated dishes in GM+6 ng/ml basic fibroblast growth factor +0.25 mg/ml Fungizone. The satellite cell cultures were observed carefully and differentially passaged to eliminate any fibroblast contamination over 5 passages. To differential passage, satellite cells were treated to a mild trypsinization and observed under a microscope until the round satellite cells were seen detaching from the plate. Because of their morphological differences, satellite cells will detach from the dish CX-4945 side effects before the flattened and larger fibroblasts. Once the satellite cells were observed detaching, the trypsin solution was immediately removed and plated to isolate the satellite cell population. To perform clonal assays, proliferating satellite cells were trypsinized and plated at 1000 cells per 10 cm dish in GM with 6 ng/mL bFGF to prevent premature differentiation. Cells were left undisturbed to minimize the formation of satellite clones and fixed with AFA at regular intervals. Myoblast clones were then immunostained for myosin heavy-chain using mouse monoclonal MF-20, biotinylated rabbit anti-mouse IgG, streptavidin and biotinylated horseradish peroxidase, followed by counterstaining with 1% methylene blue as previously described. Clones were then scored by visualization at 256/506magnification on a dissecting microscope for both number and percent differentiation. For determining initial effects of FRG1 expression in synchronized cells, proliferating iC2C12-FRG1 myoblasts either exposed to doxycycline for 24 hours previously or grown in the absence of doxycycline were synchronized by mitotic shakeoff as described. Three 10-cm dishes were tapped vigorously against a hard surface to shake off mitotic cells, incubated with gentle rocking for 20 minutes to prevent reattachment, then tapped vigorously again to further dislodge mitotic cells, and finally the medium containing mitotic cells was centrifuged and plated in a single 60-mm dish. Twelve 60-mm dishes of synchronized cells were collected and placed either in the presence or absence of doxycycline.