Cytotoxicity Assay for HT-29 Colon Cancer Cells

Human colon cancer cells HT-29 were purchased from the American Type Culture Collection (Manassas, VA). The cell line was propagated at 37°C in 5% CO₂ in RPMI 1640 medium, supplemented with fetal bovine serum (10%), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells in log phase growth were harvested by trypsinization followed by two washings to remove all traces of the enzyme. A total of 5,000 cells were seeded per well of a 96-well clear, flat-bottom plate (Microtest 96®, Falcon) and incubated overnight (37°C in 5% CO₂). Samples dissolved in DMSO were then diluted and added to the appropriate wells (concentrations: 20 μg/mL and 2 μg/mL; total volume: 100 μL; DMSO: 0.5%). The cells were incubated in the presence of test substance for 72 hours at 37°C and evaluated for viability with a commercial absorbance assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega Corp Promega) that measured viable cells. Survival percentage, based on microplate reader (Synergy Mx, BioTek) readings of absorbance at 490 nm, was expressed in percentage relative to the solvent (DMSO) control. One of the authors (Dr. Wei-Lun Chen) performed the bioassay and interpreted the results (Ren et al., 2017 (link)).