SCE Methodology for Analysis of P7 and P8
Dermal fibroblasts were treated with 26.05 μM BrdU for 72 hr followed by 0.5 μg/mL colcemid for 5 hr and then harvested. Cells were resuspended in a pre-warmed hypotonic solution (0.051 M KCl) for 20 min at 37°C, fixed with methanol and acetic acid (3:1 vol/vol), and dropped onto slides. Samples were aged for at least 3 days and then stained with 1.56 μg/mL Hoechst 33258 (Sigma, B2883). Slides were irradiated in 2× SSC with ultraviolet C (356 nm) at 0.260 J/cm2, washed with 2× SSC, and stained with 10% Giemsa in phosphate buffer.
SCE analysis of peripheral-blood samples was performed according to standard methodology in a diagnostic lab setting.27