2.7. Caco2 Cell Culture

Caco-2 cell line, derived from a human colon adenocarcinoma (American Type Culture Collection, ATCC), was used between passages 10 and 30 and cultured at 37 °C in a humidified atmosphere of CO₂/air (5/95, v/v) in Dulbecco’s MEM with Glutamax supplemented with 25 mM HEPES, 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS), 1% penicillin (1 × 10³ U/mL)-streptomycin (10 mg/mL), and 1% (v/v) non-essential amino acids (NEAA). Differentiated cells, grown to confluence for 18–21 days in 12-well plates replacing the media every 3 days (cell density of 60 × 10³ cells/cm²), were used for the experiments. For the treatments only freshly prepared and filtered (0.2 µm) HME were used. Cytotoxicity of the manna extract on Caco-2 cells was excluded by pilot studies using the Trypan Blue exclusion method and the MTT assay.

Differentiated monolayers of Caco-2 were preincubated with or without HME, at the indicated concentrations, for 1 h, and then were exposed to 25 ng/mL IL-1β for 24 h. Control cells were incubated with medium alone. After treatment, intracellular levels of ROS and GSH were cytofluorimetrically measured using the fluorescent probes 2′,7′-dichlorofluorescin diacetate (DCFDA) or 5-chloromethylfluorescein diacetate (CMFDA), respectively. Briefly, DCFDA or CMFDA (10 µM and 1 µM final concentration, respectively) were added to the medium 30 min before ending the treatment of the cells. The medium was then removed, and the cells were washed with PBS, resuspended in the same buffer and immediately analysed using flow cytometry (Epics XL^TM, Beckman Coulter, Fullerton, CA) measuring fluorescence intensity in the FL-1 fluorescence channel at an excitation wavelength of 488 nm and emission wavelength of 530 nm. At least 10,000 events per sample were evaluated.

IL-8 and IL-6 concentrations were quantified using ELISA kits according to the manufacturer’s instructions (DuoSet ELISA, R&D systems, Minneapolis, MN, USA).