Celltiter 96 cell proliferation assay kit

Cell viability assay was performed as described previously [56 (link)]. Cells were seeded on 96-well plates. After indicated stimulation, cell viability was measured by phenazine methosulfate (PMS)/3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay using a Cell Titer 96 Cell Proliferation Assay kit (Promega, Madison, USA), according to the manufacturer’s protocol. The absorbance was read at 492 nm using a microplate reader. Data are normalized to control (100%) without stimulus.

Cells were seeded at 10,000 per well in 96-well culture plates and allowed to grow for 24 hr followed by the desired treatment with increasing concentrations of the indicated agents (Enzalutamide, Abiraterone, GDC-0980, GDC-0941, LY294002 and PF-04691502) for 4 days. Viable cell densities were determined using a CellTiter 96 Cell Proliferation Assay kit (Promega). Absorbance readings at 490 nm were analyzed against the control group for each drug treatment to determine cell viability. The studies were performed in triplicates x 4 and IC₅₀ values were estimated by Calcusyn software (Biosoft, UK). For combination studies of AR inhibition plus PI3K/mTOR inhibition, an equipotent ratio was calculated to determine a combined graded combination treatment. The equipotent ratio is the ratio of the median effects resulting from the single dose treatments of AR inhibitors and PI3K/mTOR inhibitors. A control group was established for each drug treatment in six replicates. The effects of the combined treatments were determined by the combination-index (CI) and isobologram methods derived from the median-effect principle of Chou and Talalay.

The in vitro biocompatibility of the combination of Fer and SnF₂ was investigated in HGK cells using an MTS [(3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay (CellTiter 96 cell proliferation assay kit; Promega, WI, USA). HGK cells were kindly provided by the laboratory of Dana T. Graves (School of Dental Medicine, University of Pennsylvania) and were cultured in keratinocyte growth medium (Lonza, USA). To determine the cytotoxicity, HGK cells were seeded in 96-well plates at a density of 10⁴ cells per well. Cells were then incubated at 37°C in a humidified 5% CO₂ atmosphere in a cell incubator for 24 h. Afterward, old media was replaced with 100 μl of fresh media with or without Fer (1 mg of Fe/ml) and SnF₂ (250 ppm of F), or either alone, and incubated for 10 min.
After that, the media was removed, the cells were washed twice with sterile phosphate buffered saline (PBS) and 100 μl of fresh complete cell culture media was added to each well. After 24 h incubation, the cell culture media was removed, and 20 μl of MTS reagent and 100 μl of media were added to each well. After 3 h additional incubation under standard cell culture conditions, the absorbance was recorded at 490 nm using a microplate reader. The cell viability was calculated using the following formula:
$$\text{Cellviability}=\frac{{\text{A}}_{\text{490}}^{\mathit{\text{treated}}}}{{\text{A}}_{\text{490}}^{\mathit{\text{untreated}}}}\times 100\%$$

The levels of intracellular xCT were knocked down using premade siRNAs of xCT (FlexiTube siRNA, Qiagen, set 1: SI00104895, set 2: SI00104902, set 3: SI00104909, and set 4: SI00104916). Cells at 70–80% confluence were transfected with 0, 2.5, 5, 10, 20 nM of sixCT using Lipofectamine 2000 (InVitrogen, Carlsbad, CA, USA). To ensure the efficacy of sixCT, 10 nM of sixCT was transfected into xCT overexpressing stable clones and cell proliferation was measured at 48 h after the knockdown using CellTiter96 cell proliferation assay kit (Promega, Madison, WI, USA).

For the cell viability assay, an MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay was performed as described in a previous study [21 (link), 42 , 43 ], using a CellTiter 96-cell proliferation assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. In this step, 2 × 10⁴ cells were seeded on a 96-well assay plate and treated with MNPs@SiO₂(RITC) and SiO₂ NPs for 12 h. MTS solution was added to each well of the 96-well assay plate containing treated cells in 100 µL of culture medium. The assay plate was then incubated for 1 h under 5% CO₂ at 37 °C. The amount of soluble formazan produced by cellular reduction was directly measured with a plate reader at a wavelength of 490 nm. The values were normalized relative to the protein optical density value for each corresponding group.

Twenty-four hours prior to the drug treatment, cells (with a density of about 6 × 10³ per well) were seeded in the 96-well plate. Docetaxel or carboplatin were used to treat the cells using the indicated dosage for 48 h. We determined cell viability using the CellTiter96^® cell proliferation assay kit (Promega, Wisconsin, CA, USA) and followed all manufacturer instructions. GraphPad Prism software was used to calculate the IC₅₀ value of cells treated with and without docetaxel or carboplatin. All experiments were performed in triplicates and repeated thrice.

The in vitro biocompatibility of the combination of Fer and SnF₂ was investigated in HGK cells using an MTS [(3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay (CellTiter 96 cell proliferation assay kit; Promega, WI, USA). HGK cells were kindly provided by the laboratory of Dana T. Graves (School of Dental Medicine, University of Pennsylvania) and were cultured in KBM-2 medium (Lonza Group AG, Basel, Switzerland). To determine the cytotoxicity, HGK cells were seeded in 96-well plates at a density of 10⁴ cells per well. Cells were then incubated at 37 °C in a humidified 5% CO₂ atmosphere in a cell incubator for 24 h. Afterward, old media was replaced with 100 µl of fresh media with or without Fer (1 mg of Fe/ml) and SnF₂ (250 ppm of F), or either alone, and incubated for 10 min. After that, the media was removed, the cells were washed twice with sterile phosphate buffered saline (PBS) and 100 µl of fresh complete cell culture media was added to each well. After 24 h incubation, the cell culture media was removed, and 20 µl of MTS reagent and 100 µl of media were added to each well. After 3 h additional incubation under standard cell culture conditions, the absorbance was recorded at 490 nm using a microplate reader. The cell viability was calculated using the following formula: $\mathrm{Cell}\mathrm{viability}=\frac{{\mathrm{A}}_{490}^{treated}}{{\mathrm{A}}_{490}^{untreated}}\times 100\%$