Growth curves and proliferation rates of RC2.E10 cells incubated under different culture conditions. A,B, Growth curves of RC2.E10 cells incubated at 33°C in DMEM and 10% FBS (A) or at 39°C in either DMEM and 10% FBS or serum-free medium (SFM) (B). Initial plating density was 3 × 10⁴ cells per dish (60 mm cell culture dishes were used). Values represent mean ± SEM of six (A) or four (B) independent experiments. Some error bars are too small to be visible.C–F, Phase-contrast micrographs of RC2.E10 cells treated overnight with 10 μm BrdU and processed immunocytochemically with a monoclonal anti-BrdU antibody to determine nuclear incorporation. Culture conditions were as follows:C, 33°C in DMEM and 10% FBS; D, 39°C in DMEM and 10% FBS; E, 39°C in serum-free DMEM supplemented with N1 medium and sodium pyruvate (defined medium); andF, 39°C in defined medium containing 20 ng/ml basic fibroblast growth factor. G, Quantification of the percentage of BrdU-positive cells per field of vision (fov). Cells were visualized under light microscopy, and the number of immunocytochemically stained nuclei was counted. The total number of cells in the same field of vision was subsequently determined using phase-contrast microscopy. Values represent mean ± SEM of data gathered from three experiments performed in duplicate. At least 10–12 fov per dish were examined. DM, Defined medium.