Celltiter 96 aqueous bromide one solution reagent

The antitumor effects of the drugs were determined using an MTS assay. The IC₅₀ value, which is the drug concentration used to inhibit the cell proliferation by 50%, was calculated based on the results of the MTS assay. In the MTS assay, the cells were equally seeded on a 96‐well plate (2000 cells/well), and drug dilutions were added 12 hours later. The concentrations of the drugs ranged from 0.16 nmol/L to 10 μmol/L. After 3 days of medication, the cell proliferation was determined using CellTiter 96 AQueous bromide One Solution Reagent (Promega). In cases where 2 drugs were administered simultaneously as a combination therapy, we used the same concentration for both drugs. The synergism of the 2 drugs was calculated as a combination index (CI) based on the result of the MTS assay using Calcusyn software (Biosoft, Cambridge, UK). Combination therapy effects of CI < 1, CI = 1 and CI > 1 were defined as synergistic effects, additive effects and antagonistic effects, respectively.

The cell growth inhibition rate was determined using a modified MTS assay with CellTiter 96 AQueous bromide One Solution Reagent (Promega) or an MTT assay with Thiazolyl Blue Terazolium (Sigma‐Aldrich). In the MTS and MTT assays, cells were seeded in 96‐well tissue culture plates (2000 cells/well) and a 10‐cm dish (2.0 × 10⁵ cells/dish), respectively, and drug dilutions were added to each well at 12 hours after seeding. Cell growth was measured at 3 days after drug treatment. The inhibitory effects against cell growth are shown as the IC₅₀. In the MTT assay, after the drug‐containing medium was completely removed after 3 days of treatment, the cells were incubated with MTT solution (600 μL MTT and 2400 μL RPMI‐1640) for 2 hours; DMSO was then added for purple formazan solubilization. In the MTT assay, the concentrations of drugs were fixed at 500 nmol/L. For the combination treatments, the concentration of each drug was 500 nmol/L. The combination effect of the two drugs was evaluated by CI. The CI was calculated from the results of MTS assays performed for both single agent and dual medication, using Calcusyn software (Biosoft). The combination effect with CI < 1 was defined as synergistic.

The sensitivity to each drug was determined by the MTS assay. Cells were plated at a density of 3000 cells per well in 96‐well plates and treated with a preset concentration of each drug. Cell viability was assayed after 72 hours of treatment using the CellTiter 96 aqueous bromide one solution reagent (Promega). For measurement of the IC₅₀ value, eight replicate determinations were obtained in three independent experiments. Data were analyzed by nonlinear regression, using Graphpad Prism Ver. 6.0.3 (GraphPad Software).