Figure 2.

Cells that express stem cell markers survive and form spheroid structures in the absence of attachment. (a) Frequency of spheroid formation is increased in Brca1 A1.8 cells sorted for SC⁺ (CD44⁺/CD24^-) as compared with SC^- (CD24⁺/CD44^-) populations. The results shown are from long-term spheroid assay performed by limiting dilution. The black bars represent numbers of spheroids formed by CD44⁺/CD24^- sorted cells/well and the white bars represent numbers from CD24⁺/CD44^- cells ± standard deviation from six replicate wells at the end of 2 weeks in culture. The numbers on the ordinate show the number of cells plated/well. One of three independent experiments is shown. (b) Unsorted Brca1 A1.8 cells plated in the absence of attachment form spheroids, which are enriched in cells expressing stem cell markers. A1.8 were plated as single cells in low binding plates for 2 to 3 weeks, and the resulting spheroids were expanded for 4 subsequent passages, dispersed into single cell population, and stained with appropriate antibodies (as described in Materials and methods). The CD44 and CD24 markers are shown as an open histogram on the left, and filled histograms show negative controls stained with matched isotype antibody. The single stain analysis is on the right panel; the double staining is shown on the bottom. Results are presented as percentage of CD44⁺/CD24^- cells from the total population. Note the appearance of a dual population that contains CD24^-/Low cells not evident in parental cells run side-by-side with the spheroid-derived cells and illustrated for the parental A1.8 cell line in Figure 1. One representative experiment from more than three is shown here.