Apoptosis analysis was performed with flow cytometry using Annexin-V (Biovision) and propidium iodide. Annexin-V analysis by FACS on day 14 showed that most Oct4-GFP1 cells were positive for this apoptotic marker; indeed, the number of surviving cells declined thereafter.
Soft agar assay. Sorted STAP cells (Oct4-GFP-strong or -dim) and control mouse ES cells (1,000 cells per well of 96-well plate) were plated into soft ager medium(0.4% agarose) in LIF-B27 medium. After 7 days of culture, cells were dissociated and their anchorage-independent growth was quantified by fluorescent measurement with the cytoselect 96-well cell transformation assay kit (Cell Biolabs) according to the manufacturer’s protocol.
STAP論文では、 spherical coloniesを注入したと書いてあるだけです。 When the STAP conversion conditions (low pH) were applied to CD451 lymphocytes, most day-7 clusters that were large and contained more than a few dozen small cells were positive for Oct4 (although the expression level varied).
Therefore, we used only well-formed characteristic clusters (large ones) for this type of study and cut them by microknife to prepare donor cell clusters in a proper size for glass needle injection.
However, when we used ATP instead of HCl, also based on a suggestion by the authors, a few cells in a subset of cell aggregates expressed the pluripotency marker Oct3/4 at levels comparable to those in ES cells that were reproducibly detected by QPCR (Fig. 3c) and immunostaining (Fig. 4b).
On observation with fluorescent microscopy, most aggregates showed both green and red fluorescence, a sign of auto-fluorescence (Fig. 5a) although this may also include the fluorescent signal from the GOF transgene, since we detected Gfp mRNA by qPCR in these cells after culture in vitro for seven days (Fig. 3a).
Figure 7 Culture of cell aggregates in vitro. (a) The outgrowth culture of cell aggregate derived from liver cells. Liver cells were prepared from 7-days old of C57BL6 CAG-GFP Tg, treated with ATP and cultured for six days. Single cell aggregates were isolated and cultured on MEF feeder cells with medium containing KSR, ACTH and LIF adapted to the culture of ES cells. The cells continue to grow for 15 days but did not give secondary colony after passage. (b) Outgrowth culture of cell aggregates derived from liver cells. Liver cells were prepared from 4-day-old C57BL6/129 mice, treated with ATP, and cultured for six days. The cell aggregates were isolated and cultured on MEF feeder cells with medium containing FGF4 and heparin adapted to the culture of TS cells. The cells continued to grow for 11 days, but did not give rise to secondary colonies after passage.