Celltiter 96 aqueous non radioactive cell proliferation assay reagent

The cytotoxicity of NHDC and CTP on hASCs was estimated using the CellTiter 96 aqueous nonradioactive cell proliferation assay reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) (Promega, Madison, WI). hASCs at a density of 1 × 10⁴ cells/well were cultured in the presence or absence of NHDC and CTP at the concentration of 5, 10, 20, 40 μM/L in the 96-well culture plates. Various concentrations of NHDC and CTP were treated on the first and third days. After 5 days, the cell medium was replaced with 15 μL MTS reagent in 100 μL Basal medium and incubated at 37 °C with 5% CO₂ for 2 h. The absorbance at 490 nm was measured using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Cells were reverse transfected with different siRNAs used in this study in a 96-well plate for 48 h. Cell viability was then assayed using the CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay reagent (Promega).

Cell proliferation was determined using Cell Titer 96 aqueous non-radioactive cell proliferation assay reagent (Promega). The assay was performed following the manufacturer's protocol. Briefly, cells were treated with PBS or 0.6 mM VPA for seven days and equal numbers of treated HT22 (1000 cells), GL261 (2000 cells), D54 (2000 cells) and Daoy (2000 cells) cells were plated in 96-well plates. The cells plated in the 96 well plates had the respective drug or vehicle control. Cell viability was determined after incubation for 96 h by measuring the absorbance at 490 nm with a Countess II L plate reader (Life Technologies). The cells in 96-well plate were visually scanned in an inverted microscope AE30 (Motic) after 96 h of incubation, to ensure that cells were not overly confluent in some experimental arms before performing the proliferation assay. Experiments were performed in triplicate, and both average fold changes relative to controls and standard errors were calculated.

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were performed with CellTiter 96™ Aqueous Nonradioactive Cell Proliferation Assay reagent (Promega, Madison, WI) according to the manufacturer's instructions. In brief, PCA cells were plated in 96-well plates, allowed to adhere overnight and treated with the selected compounds for 72 h. Subsequently, MTS reagent was added. Absorption at 490 nm was measured after approximately 2 h using a colorimetric plate reader (Molecular Devices, Sunnyvale, CA).
To compare the antitumor effect of single-agent treatments with combination treatment, synergy was determined using CalcuSyn software (Biosoft, Cambridge, U.K.). CalcuSyn calculates a combination index (CI) at different levels of growth, using the formula for mutually nonexclusive mechanisms: (D1/Dx1) + (D2/Dx2) + (D1 × D2/Dx1 × Dx2), where D1 and D2 are the doses of drug 1 and drug 2 in combination required to produce × percentage effect, and Dx1 and Dx2 are the doses of drug 1 and drug 2 alone required to produce the same effect. Synergy levels (no synergy [CI > 0.9], moderate synergy [0.7 < CI < 0.9, +], synergy [0.3 < CI < 0.7, ++], strong synergy [0.1 < CI < 0.3, +++], very strong synergy [CI < 0.1, ++++]) were determined from CI ranges, using the Chou–Talalay method following the manufacturer's instructions 21 ,22 (link).

Cells (10,000 cells per well, 100 μl) were seeded into 96-well plates and allowed to adhere overnight. The following day drugs were added and cells were permitted to proliferate for 24 hours before the addition of 10 μl CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay reagent (Promega, Southampton, UK) as previously described^{11 (link)}. Absorbance (490 nm) readings were taken at 0, 1, 2 and 3 hours to monitor formazan formation and rates of metabolic activity were compared.

ChECs (3 × 10³ in 0.1 mL) were grown in 96-well plates (12556008; Fisher Scientific) overnight and then incubated with different concentrations of drug FTY720 (11975), FTY720 (S)-Phosphate (10006408), FTY720 (R)-Phosphate (10006407), and CYM5442 (16925; all from Cayman, Ann Arber, MI, USA), or DMSO (D8418; Sigma) in serum-containing medium for an additional 1–4 days. A cell titer 96 aqueous non-radioactive cell proliferation assay reagent (G5421; Promega, Madison, WI, USA) was added each day to a portion of the cells according to the manufacturer’s instructions. Absorbance was measured at a wavelength of 490 nm using a plate reader (Bio Tek, Santa Clara, CA, USA). The percent viability relative to control untreated cells is shown. Each experiment was repeated twice with at least two different isolations of cells.