4.3. Cell Viability Assay (MTT Assay)

Both SW620 and HT29 cells were grown in an incubator at 37 °C under 5% CO₂ condition with their respective media. After the cells were harvested, they were counted and seeded at 1 × 10⁵ cells per well in a 96-well plate overnight before being treated with DMCH. The following day, cells were treated with various concentrations of DMCH. Then, the treated cells were incubated for different time periods, which were 48 h and 72 h. Following incubation for the designated time period, 20 μL of (5 mg/mL) MTT 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (Merck, Kenilworth, NJ, USA) reagent was added into each well and then cells were incubated again for another 3 to 4 h. Next, the solution was removed and 100 μL of dimethyl sulfoxide (DMSO) was added into the wells. Afterward, the plate was read at 575 nm using a microtiter plate reader (μQuant, Bio-Tek Instrument, Winooski, VT, USA). The results were analysed as the percentage proliferation of the cells regarding the concentration of the sample treated. The following formula was used to determine the percentage of viable cells:

Percentage of viability (%) = (Absorbance of sample at 570 nm)/(Absorbance of control at 570 nm) × 100.