Characteristics of ALDH+/GD2+cells. (A) The expression of Aldehyde dehydrogenase 1 (ALDH) and GD2 on xenografted BC-P007 cells was determined by flow cytometry. (B) ALDH+/GD2+ and ALDH-/GD2- cells were sorted from the xenografted BC-P007 cells and cultured in a mammosphere condition at 1,000 cells/well of 24-well plates to mammosphere formation. Representative images of mammospheres formed from ALDH+/GD2+ (left panel) and ALDH-/GD2- (right panel) cells were shown. The distribution of ALDH and GD2 expression on the mammospheres was examined by immunofluorescent microscopy with ALDH-AF594/GD2-AF488 antibodies. Single confocal sections (C) of mammospheres stained for ALDH (red), GD2 (green) and nucleus (blue) are presented. (D) The mammosphere forming capacity of the indicated cell populations sorted from BC-P007, BC-P107 and BC-P515. The mammospheres of the indicated cell populations of these three xenografted tumor cells were counted and their mean mammosphere formation efficiency was presented as the mean ± SEM of triplicate experiments from each of the three independent xenografts. *, P <0.0001.