Recombinant human egf

HCT-8 cells (2.5 × 10⁵ per well) were seeded in the ultralow attachment 6-well plates (Corning Incorporated, Corning, NY, USA). Spheroid cells were cultivated for 6 days in the spheroid culture media (DMEM containing B27 (Life Technology, Waltham, MA, USA)), 20 ng/ml of bFGF (PEPROTECH, Rocky Hill, NJ, USA), 20 ng/ml of recombinant human EGF (Cell Signaling), and 1% antibiotics. To verify the competence of colonospheres, 6-day cultured spheroids were fixed with 4% paraformaldehyde containing 1% triton X-100 at 4 °C overnight, then fixed with 4% paraformaldehyde over 2 h. Spheroids were washed with PBS, blocked with 3% BSA, and then incubated with mouse anti-human CD44 (1:200, Cell signaling) at 4 °C overnight. The fixed spheroids were subsequently washed in PBS, incubated with Cy3-conjugated goat anti-mouse IgG (Abcam, Cambridge, UK) for 2 h at room temperature, washed in PBS, and counterstained with 100 ng/ml DAPI (absorbance at 405 nm, Sigma-Aldrich) in PBS for 10 min. Spheroids were washing with PBS 5 times for 8 min each, observed, and analyzed under a confocal laser scanning microscope (FV1000, Olympus, Nagano, Japan). To compare sphere sizes, about 200 spheroids per group were randomly selected, after which their perimeters were measured and statistically analyzed.