Sister Chromatid Exchange Analysis in Mouse ESCs and NSPCs

SCE assay was performed as previously described^{6 (link),51 (link)}. Briefly, 1 × 10⁶ mouse ESCs were cultured without feeder in ESC medium supplemented with 20 μM bromodeoxyuridine (BrdU, Sigma) for 24 h. In the final 2 h, cells were treated with 0.02 μg/mL colcemid. For SCE assay of mouse NSPCs, cells were cultured in NSPC medium adding 20 μM BrdU for 48 h. In the final 4 h, NSPCs were incubated with 0.02 μg/mL colcemid. Cells were harvested and re-suspended in hypotonic buffer (0.4% sodium citrate and 0.4% KCL) for 15 min at 37 °C. The swollen cells were fixed in freshly prepared methanol-acetic acid fixative (3:1) for 30 min and dropped onto ice-cold wet slides. The slides were dried at 65 °C overnight. The dried slides with metaphase spreads were immersed in 10 μg/mL of Hoeschst 33258 for 20 min and washed with 2 × SSC buffer (0.3 M NaCl, 0.03 M sodium citrate; pH 7.0) once. Then the slides covered with lens paper were exposed coverslip-side-up in 55 °C 2 × SSC buffer to 365 nm UV light at a distance of 1 cm for 30 min. The slides were immersed in 1 × SSC and incubated for 1 h at 50 °C. The slides were stained with 10% Giemsa solution (Gibco, 1892798) for 10 min at 37 °C. After washing with water and dried overnight at room temperature, SCEs images were captured using an Olympus confocal microscopy system.