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?0.05, **P?0.01, ***P?0.001) Among different published protocols there is considerable variability which may influence the formation efficiency and other properties of spheres [20, 37, 40]. As layed out above, we established spheroid formation from human colon cancer cells using DMEM/F12 medium supplemented with N-2, bFGF and EGF. Some of previous reports recommended the use of MEGM supplemented with B-27, bFGF, Heparin and SingleQuots (made up of insulin, recombinant epidermal growth factor (rEGF) and hydrocortisone), while some added only B-27 and rEGF. These protocols were assessed using different conditions in different cell lines but no significant difference in spheroid formation was observed in these cells [36, 38, 39]. Below are methods for troubleshooting which may help increase high colonosphere formation efficiency. First, start the experiment with low-passage cell line, and limit the number of passaging. We use CRC cell lines of up to 10C12 passages (up to 2?months of in vitro culture). Another factor is the activity of growth factors; N-2, EGF and bFGF are added to stem cell medium DMH-1 immediately before use, as these growth factors may quickly undergo degradation in the medium. Furthermore, Poly-L-lysine is usually a charge enhancer, and therefore, it can be used for coating many surfaces as it contains L- isomer for cell attachment. However, as layed out above, we have chosen to coat coverslips with poly-L-Lysine while other protocols reported coating with gelatine instead [41, 42]. One major advantage DMH-1 of using this particular protocol is usually that colonospheres are generated directly on coverslips from the beginning of the experiment; whereas other protocols generate colonospheres for 2?weeks in plates and then transfer them to the coverslips, which requires more time. However, the current protocol has a number of limitations. Because colonospheres are formed in a very small fraction, to obtain high number of colonospheres for large scale experiments, may require using a lot of expensive stem cell growth medium. Furthermore, primary colonospheres formed over a period of 10?days to 2?weeks of incubation in culture. Maybe using of new recombinant brokers and a co-culture system with colonic myofibroblasts that could promote stemness activity, can decrease the time.