Brdu labeling and detection kit 3 assay

The rate of C2C12 myoblast proliferation was assessed using the colorimetric 5-bromo-2′-deoxy-uridine (BrdU) Labeling and Detection Kit III assay (Roche Applied Science, Indianapolis, USA) modified as follows. C2C12 cells were plated at a concentration of 10⁴ cells/ml in a 6-well tissue culture plate. Twenty-four hours later, myoblasts were transfected with pFLAG-mSTARS or pFLAG-CMV4 as described above, following what myoblasts were incubated in complete DMEM (10% FBS) for 24 h. Twenty-four hours before the analysis, the BrdU label reagent and CCG-1423 or DMSO treatment were added to the medium as described above. Nucleases were used to fragment DNA and BrdU incorporation into the DNA was detected using a horseradish peroxidase conjugated anti-BrdU antibody. Following the addition of the peroxidase substrate ABTS (2,2′-azino-bis, 3-ethylbenzthiazoline-6-sulphonic acid), fluorescence was detected at 405 nm.

The rate of mouse C2C12 myoblast proliferation with and without EPO treatment was assessed using the colorimetric 5‐bromo‐2′‐deoxy‐uridine (BrdU) Labeling and Detection Kit III assay (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. Briefly, cells were plated at a concentration of 10⁴ cells/mL in a 96‐well tissue culture plate. The BrdU label reagent was added to the medium for 24 h prior to each time point analysis. Nucleases were then used to fragment DNA and BrdU incorporation into the DNA was detected using a horseradish peroxidase conjugated anti‐BrdU antibody. Following the addition of the peroxidase substrate ABTS (2,2′‐azino‐bis, 3‐ethylbenzthiazoline‐6‐sulfonic acid), fluorescence was detected at 405 nm.