White walled 96 well plate

Cells were seeded in quadruplicates at a density of 2,500–5000 cells in 100 μl DMEM per well of the white walled 96-well plates (Corning/Costar, 3917). Next day, the medium was aspirated, each well was washed with 200 μl PBS after which treatment medium was introduced. At the end of the experiment duration (48 or 72 h), relative proliferation was determined with CellTiter-Glo 2.0 Cell Viability Assay Kit (Promega, G9243) and the luminescence quantified using a SpectraMax M3 Microplate Reader.

Cells were cultured in complete media in white-walled 96-well plates (CoStar, Corning, NY) for 24 h, treated with increasing concentrations of etoposide or DMSO, and then incubated for 72 h. Cell viability was quantified using the CellTiter-Glo Assay (Promega, Madison, WI) as per the manufacturer’s instructions.

Gastric cells were washed once with Dulbecco’s PBS (DPBS; HyClone) and collected via dissociation with TrypLE Express (Gibco). Cells were pelleted by centrifugation (300 x g, 3 min, room temperature in 15-ml conical tubes (Corning), followed by 21,000 x g, 2 min, room temperature in microcentrifuge tubes). Harvested cells were resuspended in PBS (Gibco), transferred to white-walled 96-well plates (Corning), and analyzed using the GSH-Glo Assay kit (Promega) according to the manufacturer’s instructions. Intracellular GSSG and total GSH concentrations were measured using the GSH/GSSG-Glo Assay kit (Promega) according to manufacturer’s instructions. Luminescence was measured using a SpectraMax i3x Multi-Mode Microplate Reader.

To determine the viability, cytotoxicity, and late apoptosis simultaneously in 2D cell culture, ApoTox-Glo^™ Triplex assays (Promega Corporation) were performed. Cells were seeded in white-walled 96-well plates (Corning Incorporated) using 2 × 10⁴ cells per well and treated with IC₅₀ of PRMT inhibitors and dimethyl sulfoxide (DMSO) using the same concentrations and medium as controls. MUG Lucifer prim was treated with GSK 3368715 for six days and MUG Lucifer met was treated with AdOx for 72 h, as the inhibitors did not show any remarkable inhibition reversely. After incubation, 20 µl of viability/cytotoxicity reagent were added to each well and the plate was incubated for 30 min at 37 °C in a humidified atmosphere containing 5% CO₂. Fluorescence was measured at 400_Ex/505_Em for viability determination and at 485_Ex/520_Em for cytotoxicity determination using CLARIOstar microplate reader. Accordingly, the plate was incubated for 30 min at RT after adding 100 µl of Caspase 3/7 Glo reagent to each well for late apoptosis assessment. Changes in apoptosis were determined by comparing the luminescence of treated samples to vehicle controls.