Fig. 2

Serial cell sorting is essential to isolate highly pure CD133+ and CD133- populations. a Cell proliferation after seeding 10⁵ LM-MEL-62 CD133- and CD133+ cells, from an initial FACS sort. Cells stained with crystal-violet after two weeks’ growth. b Serial FACS cell sorting example of CD133+ (green), and CD133- (red) melanoma cells from primary cell line LM-MEL-34. Sort 1: Sort gates set to collect extreme 5 % of population (Sort 1 Gates), with resulting sorted populations shown (Post-sort 1 Analysis). Sort 2: Sort 1 cells cultured for ~2 weeks and CD133 expression re-analyzed (Pre-sort 2 Analysis). Cells re-sorted (Post-sort 2 Analysis). Sort 3: Sort 2 cells cultured and CD133 re-analyzed (Pre-sort 3 Analysis), then re-sorted (Post-sort 3 Analysis). Data representative of 2 replicate experiments per cell line. c Percentage CD133 surface expression by flow cytometry of CD133+ and CD133- cells from 3 primary cell lines pre- and post- 3 sorts and on day of injection. Data are representative of 2 replicate experiments +/- standard error