4 well culture chamber slides

The “stemness” of iPS cells was estimated by examining the expressions of Oct3/4, Nanog, and SSEA-4 using immunostaining. Briefly, iPS cells were cultured in 4-well chamber culture slides (Nalge Nunc International) for 5 days, and then fixed with 1% formaldehyde for 10 min. After blocking, the cells were incubated with primary antibodies against human Oct3/4, Nanog, and SSEA-4 (R&D Systems, Inc.) for 1 hr and then with the appropriate Alexa 680-conjugated secondary antibodies for 20 min. The nuclei were stained with Hoechst 33258. Staining for the expression of ALP was performed using an Alkaline Phosphatase staining kit (Cosmo Bio Co., Ltd).
The expression levels of Oct3/4 and Nanog were further examined by Western blotting, as described previously9 (link)22 (link). Briefly, total protein was purified from iPS cells, separated using SDS-PAGE gels, and then transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies against Oct3/4, Nanog, or β-actin, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences).

To detect the DNA damage, freshly purified c-kit⁺ cells were seeded on 4-well chamber culture slides (Nalge Nunc International, Roskilde, Denmark) coated with 10 μg/ml fibronectin (Invitrogen) at a density of 3 × 10⁴ cells/ml in IMDM 1640 medium supplemented with 10% fetal bovine serum (HyClone). Cells were incubated at 37°C in 5% CO₂ for 24 hours, and then fixed in 1% formaldehyde for 10 min. After blocking with 2% bovine serum albumin, the cells were reacted with anti-mouse 53BP1 antibody (Abcam), followed by a FITC-conjugated secondary antibody. The nuclei were stained with Hoechst 33258. The positively stained cells were observed under fluorescence microscopy with 200-fold magnification, and more than 200 cells were counted to calculate the percentage of cells with 53BP1 foci in the nucleus33 (link)34 (link).