Celltiter 96 non radioactive cell proliferation assay mtt kit

HepG2 cells were plated on 384-well tissue culture treated polystyrene plates at 2000 cells in 25 μl of HepG2 maintenance medium per well. After an overnight incubation at 37°C, the cells were dosed with test compounds in dimethyl sulfoxide (DMSO) and controls over wide ranges of concentrations (Table 1) and incubated for 72 h at 37°C. DMSO was at 0.5% in the final solution. The aqueous solubility of the compounds limited the range of concentrations tested. Cell viability was measured using the Promega CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) kit by adding the Dye Solution to each well and incubating for 3 h at 37°C. After incubation, the Solubilization Solution/Stop Mix was added to each well. Plates were incubated at 37°C for 1 h, mixed on a plate shaker for 10 min and then absorbance was read at 570 nm. Cell viability was determined by comparison against control cells in the presence of DMSO only and was measured in three independent replicates for each compound concentration. The IC₅₀ was calculated with GraphPad Prism using a Sigmoidal Dose-response (variable slope) algorithm. Chlorpromazine, a drug with a well-known and characterized hepatotoxicity (Frötschl et al., 2005 (link); Gandhi et al., 2013 (link); Morgan et al., 2019 (link)), was used as the positive control.

Cell proliferation assay was performed using CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) kit (Promega) according to the manufacturer’s instructions. Cells (1×10⁴cells/mL) were seeded into a 96-well plate (100 µl/well) and cultured in an incubator with 5% CO₂ at 37°C. Each well was added with 10 µl MTS solution and then incubated at 37°C for 2h. The spectrophotometric absorbance at 590 nm was measured for each sample. All the experiments were repeated 3 times in triplicates.

Vero cells were plated on 24-well plates and incubated at 37 °C and 5% CO₂ for 24 h. Prior to the addition of the compounds, media was removed. Dilutions of M and T were prepared in M199 cell culture media with a final DMSO concentration of 0.5%. Dilutions of 1, 10, 25, 50, 75, and 100 µM were added to the Vero cells in triplicate. Cells were observed every 24 h for 72 h to observe a cytopathic effect (CPE). The wells containing only M199 and M199 with 0.5% DMSO were treated as negative controls.
Cell viability of M and T in Vero cells was also tested using the CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) kit (G4000, Promega, WI, USA) according to the manufacturer’s instructions.

Cytotoxicity was performed using the CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) kit as manufacturer’s recommendations (Promega, Madison, WI, USA). Briefly, 1 × 10⁴ HESCs/well were seeded onto 96 well plates and incubated for 24 hr. After removal of supernatant, the plates were incubated with mixed solution (new media 100 μL and MTT solution 10 μL) for 4 h at 37 °C in a carbon dioxide incubator. Solubilization and stop solution mixture was added. The dark blue formazan product was quantified using a microplate reader at 570 nm (with a 690 nm reference filter; Molecular Device, Sunnyvale, CA, USA).

Cell viability was assessed using a CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) kit (Promega, Madison, WI, USA), based on the reduction of MTT into formazan dye by the action of mitochondrial enzymes. Briefly, HL-1 cells were seeded in 96-well plates at 1 × 10⁴ cells/well and incubated overnight at 37 °C in 5% CO₂. The cells were treated with indicated concentrations (0, 10, 100, 1000, and 5000 nM) of Ang II for 24 h. The absorbance of each well was measured at 570 nm.

After 24 h of incubation with the
sample of the protein aggregates, the cells were stained using CellTiter
96 nonradioactive cell proliferation assay (MTT) kit (Promega, Madison,
Wisconsin). Cells were incubated with the dye for 4 h at 37 °C
under 5% CO₂. Next, solubilization solution (Promega, Madison,
Wisconsin) was added and incubated for 1 h at 37 °C under 5%
CO₂. Absorption measurements were made in a plate reader
(Tecan, Männedorf, Switzerland) at 570 nm. Every well was measured
25 times in different locations.