Fig. 7.

Comparison of BrdU strand-substitution conditions between the protocol used in this study (1.25/200 μM BrdU/dCTP, 7 h, with 200-μM dCTP medium substitution persisting for another day) and the protocol used by Falconer et al. (40 μM BrdU for 16 h, no dCTP). a FACS-plot compilation of a typical experiment. We gated for live cells (bottom right), dead cells (top left), and counting beads (top corner), which were added as an internal control. Cell preparations were diluted 1:2 prior to 25-h analysis. Our analysis revealed subtle differences in live/dead cell ratios immediately after BrdU treatment (1-h samples). One day later (25 h), the cytotoxic effects of BrdU, particularly in the 40-μM sample, are evident. The fraction of live cells is reduced from ~81 to ~48 %, the number of dead cells is increased by ~70 %. The fraction of counting beads has roughly quadrupled (~6 vs. ~25 %), indicating reduced overall cell numbers. BrdU/dCTP-treated samples, in contrast, are much less affected. b Summary of development of live cell ratios between 1 and 25 h time-points. Average of three experiments normalized to counting beads and control cells, with error bars showing standard deviations. Whereas ~80 % of control live cells can be detected in the 1.25/200-μM samples, 40 μM BrdU reduces this rate to ~20 %. c Histogram plots for stem cell surface marker SSEA-1 expression assessed by flow cytometry, 25 h after BrdU was removed from the culture medium. We gated on live cells based on forward and side scatter. Whereas control and 1.25/200-μM samples show highly similar expression profiles, the variation of SSEA-1 expression is much greater in 40-μM-treated samples, and the mean expression is lowered. d We gated on cells exhibiting high SSEA-1 expression and summarized three staining experiments. Error bars show standard deviations