Spheroids/colonospheres display increased levels of CSC markers. A. Representative scattered plots of flow cytometry analyses of CD44 positive cells in different colon cancer cells: Figures in the left panel depict the parental colon cancer cells stained with PerCP-Cy5 mouse IgG2b (Isotype), whereas those in the right panel, show the cells treated with CD44 conjugated PerCP-Cy5 mouse anti-human antibodies. Values represent percentage of cells that are positive for CD44-PerCP-Cy5. B. Photomicrographs showing the integrity and viability of colonospheres (HCT-116) using acridine orange-ethidium bromide (AO-EtBr) staining and immunofluorescence staining depicting the membrane bound expression of putative CSC marker EpCAM (Upper Panel); and flow cytometric analysis showing the proportion of CD44 positive cells in HCT-116 colonospheres-derived cells. Corresponding isotype for CD44 antibodies is shown (Lower Panel).