Celltiter 96 aqueous one solution mts solution

Cell proliferation was determined using the MTS assay as reported by Kong et al. [62 (link)]. Briefly, 5 × 10³ cells were seeded onto 96-well plates overnight. Next, cells were treated with 50 μg/mL of peptide B11 or 50 μg/mL 5-fluorouracil (5-FU) as a positive control, with PBS (0.01 M, pH 7.4) used as the negative control. After 24 h, 20 µL/well of CellTiter 96 AQ_ueous One Solution (MTS) solution (Promega, Madison, WI, USA) was added and incubated at 37 °C for 3 h. Optical density (OD) was measured using a microplate reader (BioTek, Winooski, VT, USA) at 490 nm. The viability rate was calculated as, cell viability% = OD_B11/OD_PBS × 100%. Triplicate samples were analyzed, and data were represented as means ± standard error (SD). Experiments were repeated at least three times and the p-values were determined using Student’s t-test.

Cells were cultured (2000–4000 cells/well) on microtiter culture plates and treated with various concentrations of compounds for 72 h. At assay endpoint, cells were incubated with CellTiter 96^® AQ_ueous One Solution (MTS) solution (Promega) at 37 °C for 3 h. Absorbance was measured at 490 nm. All data points were set up with three replicates for each experiment. Percent cell proliferation was calculated relative to DMSO control‐treated cells.
The sigmoidal dose–response curve fitting for belinostat and the half‐maximum inhibitory concentration (IC₅₀) were calculated by graphpad prism software (GraphPad Software, La Jolla, CA, USA). Combination index (CI) for belinostat and cisplatin was calculated based on Loewe additivity equation (Chou and Talalay, 1984), using the drug concentrations that resulted in the inhibition of cell viability between 50% and 80%, and tabulated at ED_50‐80.

CT-26 and RAW 264.7 cells were cultured (3 × 10³ cells/well) in a 96-well plate. After 24 hours, RA, CHO, NP3-(RA)-CHO, NP4-(RA), NP8-CHO, and anti-IL-10R inhibitor were added at three different concentrations as selected in a pilot study, such as: (i.e., RA: 0.5 µM, 1 µM, and 5 µM; CHO: 1 µg/ml, 5 µg/ml, and 10 µg/ml; NP3: 1 µg/ml, 2.5 µg/ml, and 5 µg/ml; NP4: 1 µg/ml, 2.5 µg/ml, and 5 µg/ml; NP8: 1 µg/ml, 2.5 µg/ml, and 5 µg/ml; abIL-10R: 0.6 µg/ml, 1.2 µg/ml, and 2.4 µg/ml). All compounds were maintained in the dark at -20°C and dissolved in PBS. After another 24 hours and 48 hours, 20 μl/well of CellTiter 96 Aqueous One Solution (MTS) solution (Promega, Madison, WI, USA) was added and incubated with 5 % CO2 at 37°C for 3 hours. All experiments were carried out in triplicate, with the average of the duplicates shown. 25 % DMSO was utilized as a positive control (“death”), and culture media without medication was employed as a negative control (“live”). Absorbance was measured at 490 nm.