Magnetically sorted CD133(+) cells exhibit markers of melanoma initiating cells. BAK cells were separated by MACS and stained for CD133 positivity (a) using CaCo-2 and 1205LU cells as positive and negative controls, respectively (not shown). CD133 positivity was then quantified after imaging with a fluorescence microscope, as well as by flow cytometry (b). (c) RNA from MACS-sorted CD133(+) and CD133(-) BAK cells was subjected to RT-PCR (left) and qRT-PCR (right) analyses for CD133, using GAPDH as a control. (d) Immunofluorescent staining reveals expression of CD133 and stem cell marker Oct 3/4 in CD133(+), but not CD133(-) cells. (e) Additional cancer stem cell markers Nestin and ABCB5 are coexpressed with CD133 and Oct 3/4, using GAPDH as loading control to determine expression levels. (f) Cells were double stained using antibodies specific for ABCB5 and CD133. Cells positive for ABCB5 were quantified in CD133(+) vs. CD133(-) populations. (g) Melanosphere formation in CD133(+) (top), but not CD133(-) cells (bottom).