Automacs pro cell sorter

Primary human myoblasts from a young, lean donor were used in these experiments and obtained through the Center for Muscle Biology Healthy Muscle Bank. In order to obtain a relatively pure (>90%) myoblast culture, following tissue digest with collagenase and dispase, cells were subjected to magnetic antibody cell sorting (MACS) using anti-CD56 microbeads and an Automacs Pro cell sorter (Miltenyi Biotec, Auburn, CA). The sorted cells were expanded in growth media consisting of Ham’s F-10 (Fisher Scientific, Waltham, MA), 20% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA), 1% penicillin/streptomycin (Fisher Scientific), and 10ng/ml basic fibroblast growth factor (bFGF) (Peprotech, Rocky Hill, NJ). To mimic the obese environment, myoblasts were cultured in growth media supplemented with 25 mM glucose, 100 nM insulin, and 250 Mm palmitate for 24 hours. For irradiation experiments, myoblasts were exposed to 5 Gy irradiation in a Mark I-68 137Cesium γ-irradiator (J.L Shepherd and Associates, City, State) on a rotating platform. At the designated time points (30 minutes, 2 hours, and 8 hours post-irradiation), myoblasts were fixed in 4% paraformaldehyde (PFA) and centrifuged at 1000 RPM onto slides using a cytospin (Cytospin 4; ThermoFisher, Waltham, MA). Slides were dried for 1 hour before immunocytochemical analyses.