Spectra190

Cell proliferation was determined by thiazolyl blue tetrazolium bromide (MTT, Sigma) assay. Viable cells convert MTT to insoluble formazan crystals. Cells were seeded at a density of 1 × 10⁴ cells per well in 96-well plates. The cells were treated with Genipin for 24 h and subsequently with MTT solution for 4 h at 37 °C. The absorbance at 595 nm was measured using a microplate reader (SPECTRA190, Molecular Devices, Sunnydale, CA, USA).

Cell proliferation was determined by the WST assay using EZ-CyTox Cell Viability, Proliferation, Cytotoxicity Assay kit (DoGEN, Daeil Lab Service Co. Ltd, Seoul, South Korea). Cells were seeded at a density of 1 × 10⁴ cells per well in 96-well plates. Cells were then treated with CBD for 24 h and then treated with WST-1 for 3 h at 37 °C. Absorbance at 450 nm was measured using a microplate reader (SPECTRA190, Molecular Devices, Sunnydale, CA, USA).

Cell viability was measured using the Cell Viability Assay Kit (EZ-Cytox, DOGEN, Daejeon, Korea). Human CRC HCT116, DLD-1, HT29, and SW620 cells, and normal colon FHC and CCD-18Co cells (1 × 10⁴ cells per well) were seeded on 96-well plates and then treated as described in the Results section. The cells were then incubated with the EZ-Cytox reagent and incubated for 2 h at 37 °C in an atmosphere of 5% CO₂. Absorbance at 450 nm was determined using a microplate reader (SPECTRA190, Molecular Devices, Sunnydale, CA, USA).
To detect the translocation of phosphatidylserine, a marker of apoptosis, from the inner to the outer leaflet of the plasma membrane, the cells were stained with Annexin V according to the manufacturer’s protocol for the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA). Flow cytometry was performed using an Accuri C6 flow cytometer (BD Biosciences).