Figure 1. RB side populations (SPs) detected by flow cytometry

Side populations, characteristic of a stem cell phenotype, are identified by verapamil-sensitive exclusion of the Hoechst 33342 fluorescent dye. In this experiment, cell suspensions were incubated with Hoechst 33342 dye and analyzed by flow cytometry as described in Methods. Cancer stem cells would be expected to exclude the dye, except in the presence of verapamil, a drug that can abolish the dye excluding properties of the ABCG2 transporter. The fluorescence intensities of each cell in a population of at least 10,000 are plotted (Hoechst blue [y-axis] versus Hoechst red [x-axis]). The axes represent fluorescence intensities. The side population region was defined on the basis of fluorescence emission in both red and blue wavelengths. Boxes were drawn (labeled P4) to indicate verapamil-sensitive, Hoechst-excluding side populations. Both WERI-Rb27 and fresh SV40 βLH-T-Ag mouse RB tumors were found to contain side populations, and the percentage of cells in the boxes (as a percentage of the total number of live cells) is given in each plot. Cells within these P4 boxes are putative cancer stem cells.