The apoptosis and the proliferation on committed neuroblast cells. (A) The induction of Sox1-GFP+ cells from 46C cells were detected by flow cytometry under the SFEB and SFEB/FGF1 condition. (B) The differentiating ES cells were labeled with cleaved caspase-3 (red), which detects the cleaved fragment of caspase-3 (17/19 kDa), in Sox1/GFP+ cells on differentiating day 4. (C, D) Proliferating GFP+ cells were marked with the nuclear staining of ki67 on day 4. (E) Total apoptotic cells, characterized with Annexin-V labeling, were estimated by flow cytometry after FGF and/or z-VAD-fmk, a membrane-permeable pan-caspase inhibitor, from day 1 to day 4. Culture media were changed every day. (F) Total cell numbers were counted in triplicate using trypan blue exclusion at indicated times.