Low adherent 6 well plates

A375 and B16-F10 CICs were isolated based on ALDH activity as previously described [37 (link)]. Gates for each sample were based upon N, N-diethylaminobenzaldehyde (DEAB) controls for each cell line. Sorted cells were cultured in low-adherent 6-well plates (Corning) in serum-free media at a density of 1 × 10³cells/mL. Cultures were grown for up to 14 days and treated with fresh media containing either 100 μM Lunasin or vehicle (PB) twice per week. Oncospheres (> 100 μm) were counted and imaged using an EVOS light microscope (Life Technologies) and images were analyzed using Image-J software (NIH) as described [37 (link)].

CRC-derived CSCs were made into single-cell suspension and seeded in low-adsorption six-well plates at 1 × 10⁴/mL, cultured in serum-free medium, and treated with AT7867. The cells treated with DMSO were used as a control group. After 5 days of incubation in a 37°C incubator, pictures were counted under an inverted microscope. CSCs were treated with different concentrations of AT7867. After 72 hours, cells from each group were collected and digested into single cells. Subsequently, for each group, 1 × 10⁴ viable cells per well were counted and seeded in low-adherent 6-well plates (Corning, USA) in serum-free conditioned medium without AT7867. After 5 days of incubation in a 37°C incubator, pictures were counted under an inverted microscope.