IGF-I Stimulated DNA Synthesis Assay

DNA synthesis was assessed by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into DNA [13 (link)]. Briefly, cells were seeded in 96-well culture plates (1200 cells/well), and maintained in medium containing varying doses of IGF-I for 24h at 37° C. BrdU or phosphate buffer saline (as background control) was added to each well during the final 8h of treatment. After fixation, cells were incubated with anti-BrdU antibody for 1h at room temperature, and with substrate solution for 15 min in the dark at room temperature. The signals were quantitated using a spectrophotometric plate reader set at wavelength of 450/540 nm. To examine if high dose IGF-I also activated the other signaling pathways to stimulate DNA synthesis, cells were treated with IGF-I and AG1024 (Sigma Aldrich, MO, USA) concomitantly.