Materials and Methods
Animals
The control and LSN chickens used in the current study were from stocks maintained at The Ohio State University Poultry Genetics Center. As described in Velleman et al. (1993), LSN birds exhibit reduced pectoral muscle function in an "exhaustion score" test (the ability of the animal to right itself from a supine position). In addition, LSN birds late in embryonic development have elevated levels of TGF-β-1 and TGF-β-2 (Velleman and Coy, 1998), increased decorin proteoglycan levels, and posthatch animals are characterized by elevated collagen crosslinking (Velleman et al., 1996), and abnormal sarcomere structure (Velleman et al., 1997).
Satellite Cell Isolation, Culture, and Protein Extraction
The isolation of control and LSN satellite cells used in the present study was previously described in Li et al. (1997). Control and LSN satellite cells were cultured 7 times. Satellite cells were grown in 35 mm gelatin-coated plates (Corning Inc., Corning, NY) at a cell density of 42,000 cells/well for the control and 35,000 cells/well for the LSN and allowed to proliferate for 96 hours. These cell densities were used so the two satellite cell lines would reach approximately 65% confluency at the same time. During cell proliferation, satellite cells were cultured in McCoy's 5A (Life Technologies Inc., Grand Island, NY) + 10% chicken serum (Sigma Chemical Co., St. Louis, MO)+5% horse serum (PePro Tech, Rocky Hill, NJ) + 20 ng/mL fibroblast growth factor (PePro Tech, Rocky Hill, NJ). After the cells reached 65% confluency, differentiation and fusion were induced by low serum containing medium consisting of Dulbecco's Modified Eagle Medium (PePro Tech, Rocky Hill, NJ)+ 3% horse serum + 0.01 mg/mL porcine gelatin (Costar Corp., Cambridge, MA) + 1.0mg/mL bovine serum albumen (Costar Corp., Cambridge, MA).
At 24-hour intervals during proliferation and 12-hour intervals during differentiation, cell culture wells were sampled. Cells were removed from the culture wells by gentle scraping with a cell lifter (Costar Corp., Cambridge, MA) in HEPES buffer pH 4.2 (Costar Corp., Cambridge, MA) and transferred to a Dounce homogenizer. The samples were then dialyzed and protein concentrations were measured by the Bradford (1976) method.
Measurement of β1 Integrin Expression
Indirect ELISA analysis was performed to measure relative levels of β1 integrin. In brief, 100 μL of carbonate-bicarbonate buffer (15 mM Na2CO3 and 34 mM NaHCO3, pH 9.8) containing 10 mg of total protein from the satellite cell extracts were pipetted into duplicate wells of Falcon Pro-Bind 96-well microtiter plates (Becton Dickinson Labware, Lincoln Park, NJ) and incubated overnight at 37oC. Following incubation, the wells were washed twice with PBS-Tween 20 (PBS-T) (137 mM NaCl, 8 mM Na2HPO4.7H2 O, 1 mM KH2PO4, 3 mM KCl, and 0.05% Tween-20, pH 7.4) and once with distilled H2O. Then 100 μL of a 1:500 dilution mouse monoclonal β1 integrin antibody CSAT (Developmental Studies Hybridoma Bank, Iowa City, IA) (Neff et al., 1982) in PBS-T was added to each well and incubated at 37oC for 2 hours. After incubation with the primary antibody, the wells were washed three times with PBS-T and twice with distilled H2O. To each well, 100 μL of a 1:30,000 dilution of alkaline phosphatase conjugated goat anti-mouse secondary antibody (Costar Corp., Cambridge, MA; Chemicon Intl., Temecula, CA) in PBS-T was added and incubated at 37oC for 2 hours. Prior to color development, the wells were washed three times with PBS-T and twice with distilled H2O. One hundred μL of p-nitrophenyl phosphate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was then added to each well, and incubated in the dark for 30 minutes or until optimum color was achieved. The optical density (OD) was read at 405 nm with a Dynex Revelation microplate reader (Dynex Technologies, Inc., Chantilly, VA).