Figure 3. Cell proliferation in wild type and Hfe^−/− RPE cells.

(A) Giemsa staining or colony formation assay in a 12-well plate for wild type and Hfe^−/− RPE cells. 8,000 cells per well (left two columns) or 4000 cells per well (right two columns) were seeded in triplicates. The cells were cultured for 12 days, with medium change every 2 days. Cells were then fixed with methanol for 30 min followed by staining with KayroMax Giemsa for 1 h. (B) Wild type and Hfe^−/− RPE cells were seeded at varying cell density in a 12-well plate and cultured for 12 days. Medium was changed every 2 days. On the 12^th day, cells were fixed with methanol and than stained with KayroMax Giemsa. The contents of the wells were solubilized and the dye released was quantified. n = 6; *, p < 0.001. (C) Wild type and Hfe^−/− RPE cells were seeded at a density of 10,000 cells per well and cultured for varying time periods. At the end of the indicated time period, cells were stained with Giemsa, and the dye quantified as described above. n = 6; *, p < 0.001.