Moreover, freshly prepared medium is added to the colonosphere tradition every 3C4?days, therefore there is possibility to lose the colonospheres formed while changing press, since colonospheres are unattached floating spheroid colonies. defined medium. Consequently, great efforts have been paid to improve colonosphere forming assay like a preclinical model to study tumor biology and to conduct drug testing in cancer study. The 3D-colonosphere tradition model may also represent in vivo conditions for the spontaneous aggregation of cancer cells in spheroids. This protocol explains the development of an enrichment/culture assay using CRC-CSCs to facilitate colorectal cancer research through immunofluorescence staining of colonospheres. We have developed colonospheres from HCT116 CRC cell line to compare and link DMH-1 CRC-CSC markers to the NANOG expression level using an immunofluorescence assay. Our data also show that this immunostaining assay of colonosphere is usually a useful method to explore the role and dynamics of CRC-CSCs division between self-renewal and cell lineage differentiation of cancer cells. In theory, this method is applicable to a variety of primary cells and cell lines of epithelial origin. Furthermore, this protocol may also allow screening of libraries of compounds to identify bona fide CRC-CSC differentiation inducers. =250?m. b Whole cell lysates isolated from HCT116 cells (GFP and GFP/NANOG) were Western blotted using antibodies against NANOG and the loading control -actin The colonospheres formed typical circular structure (Fig. ?(Fig.2a)2a) and within a single CACNG1 spheroid, the cells appeared fused together resembling a solid cellular cluster making it hard to distinguish as individual cells [36, 37]. Moreover, the size of spheroids ranges from less than 50?m to 250?m (Fig. ?(Fig.3)3) [38, 39]. Next, the influence of NANOG overexpression around the efficiency of colonosphere formation was evaluated and compared with HCT116-GFP cells and GFP/NANOG cells, which exhibited an increase in spheroid formation by 14C17?%, as shown in Fig. ?Fig.33c. Open in a separate windows Fig. 3 Growth of HCT116 colonospheres (GFP and GFP/NANOG) under 3D culture in spheroid-medium prior to immunofluorescence staining. Colonosphere formation is analyzed after 2?weeks; (a) HCT116 GFP cell line and (b) HCT116 GFP/NANOG cell line. =250?m, 10 magnification. c Quantitative data showing the growth of colonosphere formation efficiency in GFP/NANOG versus GFP cells. Data represent mean??SD test was used to calculate values (*=10?m, 40 magnification. Data represent mean??SD test was used to calculate values (*P?P?P?