Cell culture experiments using Caco-2 cell lines

Human Caco-2 cell lines (HTB-37; American Type Culture Collection, Rockville, Maryland, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, New York, USA), supplemented with 1% non-essential amino acids, 1% L-glutamine, 20% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin and 100 μg/mL of streptomycin in 50 cm² plastic flasks (JET Bio-Filtration Products; Guangzhou, China). Cells were grown in a humidified atmosphere of 5% CO₂ at 37 °C and the culture medium was replaced every 2 to 3 days. Cells were sub-cultured after reaching approximately 80% confluence, and used between passages 18 and 24 in all experiments.

Caco-2 cells were grown to confluence in 96 well microplates under the conditions detailed above. The culture medium was removed and replaced with fresh medium containing 25% (v/v) of the digests diluted with culture medium. Cells were cultured for 24 h, the culture medium was then removed and the Caco-2 cells were washed with fresh culture medium without FBS. The induction of oxidative stress was carried out by exposing Caco-2 cells to 500 μM hydrogen peroxide (H₂O₂) for 1 h. The untreated cells were taken as “control” whereas the untreated cells after addition of H₂O₂ were referred as “control + H₂O₂”. Four independent trials were conducted to determine the cell viability and lactate dehydrogenase membrane release after digest supplementation.