Dimethyl sulfoxide (dmso)

At 7 dpi, cells were washed three times with PBS, supplemented with 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT, Sigma, St. Louis, MO, USA) in culture media, and incubated for 4 h in the dark. Then, the supernatant was discarded, and 100 µL of dimethyl sulfoxide (DMSO, Sigma, Marlborough, MA, USA) was added. Subsequently, the DMSO was transferred to a new 96-well plate, and absorbance was measured at 570 nm in a microplate reader (InfinitePro 200, TECAN, Männedorf, Switzerland). The cell viability percentage was determined as a function of the absorbance of non-infected control cells.

Caco-2 cell viability was assessed by using a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously reported [63 (link),64 (link)]. Briefly, viable cells (2.5 × 10⁴ cells/well) were seeded and grown for 24 h in a complete medium into a sterile 96-well cell culture cluster (Corning, Sigma-Aldrich, St. Louis, MO, USA) and maintained at 37 °C in a humidified atmosphere with 5% CO₂. Subsequently, the cells were exposed to crude milks and their extracts with the total and the free lipid fractions at the concentrations of 25 µg/mL and stimulated with 1 µg/mL of lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA) for 48 h. For raw milks, the same volume used for their total lipid fraction was added. At the end of the LPS stimulation, the culture medium was replaced by a solution of 0.5 mg/mL MTT (Sigma-Aldrich, St. Louis, MO, USA) in PBS. After 2 h of incubation at 37 °C in 5% CO₂, the supernatant was carefully removed from each well, and the formazan crystals were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). The absorbance values were measured at 570 nm using a TECAN M1000 pro using DMSO medium as blank solution. The cell viability was calculated according to the following formula: % cell viability = [optical density (OD) of tested compound/medium OD of control cells] × 100.

The cells were seeded onto 96-well culture plates at density of 3×10⁵ cells/well in 100 μL. For CA or Berberine treatment, different concentrations of CA (2.5, 5.0, 10 nmol/L) or Berberine (5, 10, 25, 50, 100 μg/mL) or culture medium containing 0.01% of DMSO, used as a vehicle control, were added to the culture medium and incubated with the cells for 12 hr. Crystal violet and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays [25] (link) were used to detect cell viability and metabolism. MTT, dissolved in phosphate-buffered saline (PBS) at 5 mg/mL, was added to the cells and incubated for 6 hr at 37°C, and the cells were lysed with DMSO before being read at 570 nm (Tecan, Salzburg, Austria). For crystal violet assay, 0.2% crystal violet in PBS was added and incubated with cells for 2–3 min, then 1% SDS was added and the color was read at 570 nm. All assays were performed in octuple repeats.