In vitro toxicology assay kit

Subconfluent conditions: Approximately 5 x 10⁴ HFF cells were plated into each well of a 24‐well dish and exposed to D10+ media alone or D10+ with DMSO, combretastatin A4 or parabulin for 3 days. The plates were processed with the In Vitro Toxicology Assay Kit, MTT based (Sigma‐Aldrich, TOX1), and A₅₇₀ was measured in a SpectraMax I3X plate reader. Exported data were analyzed using Microsoft Excel. Confluent conditions: Approximately 5 × 10⁴ HFF cells were plated into each well of a 24‐well dish and grown to D10+ media alone. Once the cells were confluent, they were gently rinsed with PBS to remove the media and then D10+ with DMSO or parabulin was added to each well. The plates were processed with the In Vitro Toxicology Assay Kit, MTT based (Sigma‐Aldrich, TOX1), and A570 was measured in a SpectraMax I3X plate reader. Exported data were analyzed using Microsoft Excel.

The In Vitro Toxicology Assay Kit (Sigma-Aldrich) was used to perform a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay to measure the cytotoxicity of the indicated compounds according to the manufacturer’s protocol. Briefly, NTCP-HepG2 cells were incubated with 100 µL of the indicated concentrations of the respective compound solved in HGM over 6 h at 37 °C. After 6 h, the medium was replaced by inhibitor-free HGM and cells were cultured for additional 24 h. Then medium was removed and 100 µL DMEM containing 0.5 mg/mL MTT were added and cells were incubated for 1 h at 37 °C. Finally, the medium was replaced by 100 µL isopropyl alcohol (Sigma-Aldrich) and samples were measured by ELISA reader (GloMax-Multi Detection System, Promega, Madison, WI, USA).

Cell viability was assessed using the in vitro toxicology assay kit (Sigma‐Aldrich) based on MTT and conducted according to the instructions.20, 21 At the end of the experiment, the medium was removed, and the cells were washed with 1 × PBS. Then, corresponding reagents (containing MTT) were added to each well. Cells were subsequently incubated for another 4 hours at 37°C. The medium was then abandoned, and 150 μL dimethyl sulfoxide (DMSO) was added to each well which was then shaken for 10 minutes at room temperature to completely dissolve the blue‐purple precipitate from the MTT. The OD value of each well was measured using a multimode plate reader (PerkinElmer Victor x3) at 570 nm. The values are expressed as percentages of low‐glucose group (LG) control values. The experiment was repeated five times.