Mouse anti human cd44

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.

Human colon cancer cells, LoVo and SW48, were purchased from ATCC (Manassas,VA) and cultured in DMEM media (Invitrogen, CA, USA) supplemented with 10% FBS (Gibco, NY, USA) in a 37°C humidified incubator with an atmosphere of 5% CO₂ and 95% air.
The antibodies used in the present study included: mouse anti human CEA (clone: Ab-3; Thermo Scientific, MA, USA), anti-biotin microbeads (Miltenyi Biotec, CA, USA), mouse anti human Epcam-APC (clone: HEA-125; Miltenyi Biotec, CA, USA), mouse anti human CD44 (clone: 156-3C11, Cell Signal Technology, USA), mouse anti-biotin FITC (clone: Bio3-18E7; Miltenyi Biotec, CA, USA), Alexa Flour 488 conjugated to goat anti mouse IgG (Jackson ImmunoResearch Laboratories, PA, USA), rabbit anti human cytokeratin 20 (clone: EPR1622Y; Abcam, CA, USA), mouse anti human GAPDH (Santa Cruz Biotechnology, CA, USA), rabbit anti human IGF1R (1159–1163, Abcam, CA, USA), goat anti human IGF-1 (Abcam, CA, USA).