2.6. CaCo-2 Culture and Cytotoxicity Assay

VAL and 4-HBA cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on colorectal adenocarcinoma cell line Caco-2 (ATCC^® HTB-37™, ATCC, Manassas, VA, USA). Compounds’ stock solutions were prepared in dimethyl sulfoxide (DMSO), diluted in complete medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1 mM sodium pyruvate) and filtered on 0.22 µm filters. The concentrations tested were 245, 100, 30, 10 µM for VAL and 36, 18, 3.5, 1 µM for 4-HBA. DMSO 0.1% (the highest concentration in the dilutions) was added as control. Briefly, 1 × 10⁴ Caco-2 cells/well were seeded in a 96-well plate and treated with serial dilutions of VAL and 4-HBA for 24 h. The MTT solution (0.5 mg/mL) was then added to each well and incubated for 3–4 h. Formazan crystals were solubilized with 100 µL/well of lysis buffer (8 mM HCl and 0.5% NP-40 in DMSO) and the absorbance measured at 575 nm in a microplate reader (Power Wave HT, Biotek, Bad Friedrichshall, Germany). The cell viability was calculated by the following formula:

Three independent experiments were performed with four technical replicates for each condition.