Clonogenic and sphere forming activity of ALDH ^high cells.

<p>A) ALDH^high and ALDH^low TC71 cells were plated in soft agar at 1000 cells/well. Data from 3 independent experiments represent average colony count/well after 14 days. B) ALDH^high and ALDH^low TC71 cells were plated in Mesencult at 1000 cells/well for 7 days. Data shown is the average sphere count from a representative experiment performed in triplicate wells. C) Average sphere count from ALDH^high and ALDH^low cells isolated from an ESFT xenograft. 3000 cell/well in triplicate wells were cultured in Mesencult for 14 days. Bars = SEM (D) ALDH^high and ALDH^low cells isolated from a second ESFT xenograft, this one with contaminating murine cells excluded as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013943#pone-0013943-g003" target="_blank">Figure 3</a> were cultured in Mesencult for 14 days in triplicate wells with 3000 cells/well. Bars = SEM. (E) MHH-ES cells were incubated with the Aldefluor reagent followed by PE-labeled anti-CD133 and then were sorted for ALDH and CD133 expression simultaneously. Only a small number of CD133-positive cells are seen, and these are evenly distributed across the ALDH spectrum (gate R3). (F) CD133-positive cells, ALDH^high cells, and control cells (CD133-negative, ALDH^low) were plated in Mesencult at 1000 cells/well for 7 days. Data shown is the average sphere count from a representative experiment performed in triplicate wells. Bars = SEM. Each experiment was performed a minimum of three times. In each panel, the p value shows the significance of the difference between the indicated populations, as determined by a one-tailed t test.