Melanospheres contain a small subpopulation of quiescent/slow-cycling GFPhighlabel-retaining cells (LRCs) with a melanoma stem cell phenotype. A. Representative melanospheres (left panel, scale bar = 50 μm) formed by HBL-H2BGFP cells dividing at different rates, as reflected by differences in the GFP fluorescence intensity between dissociated melanosphere cells (right panel, scale bar = 20 μm). B. GFPhigh melanosphere HBL cells cycle slower and incorporate less EdU than their GFPlow counterparts. C. These GFPhigh cells overexpress stem cell surface markers. Histogram illustrating the ratio of expression of each marker in GFPhigh cells to their own GFPlow controls, which were set at “1” and marked by the red interrupted line. D. Representative histograms of flow cytometry data for the surface ABCB5 marker in melanosphere HBL-H2BGFP cells. Upper histogram illustrates GFP (green) fluorescence distribution in total population. R1 is a region encompassing GFP negative (black lines) and GFPlow subset and R2 GFPhigh subpopulation (shaded). Lower histograms show ABCB5 distribution between GFPlow and GFPhigh (shaded) subpopulations. R3 is a boundary drawn around ABCB5high cells within GFPlow subset and R4 within GFPhigh subpopulation. Numbers indicate percentage of cells with a particular phenotype.