Celltiter 96 aqueous one solution reagent assay

COLO320 cells (American Type Culture Collection; ATCC, VA, USA) were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA) medium supplemented with an antibiotic-antimycotic cocktail (100 U/mL penicillin and 100 μg/mL streptomycin; Life Technologies, Carlsbad, CA) and 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) under a humidified atmosphere of 5% CO₂ in air at 37°C. Cells ( 0.5–1.0×10⁶) were seeded into 25 cm² culture flasks (Eppendorf AG, Hamburg, Germany) and passaged at 70–80% confluency. To assess the effect of puromycin on cell viability, 0.02–0.1 × 10⁵ cells were seeded in RPMI containing 10% FBS (100 μL/well) in a 96-well cell culture plate (Eppendorf AG, Hamburg, Germany). All cultures were performed in triplicate. After 12 h, the medium was replaced with RPMI 1640 medium with 10% FBS that contained 0–20 μg/mL puromycin (Life Technologies, Carlsbad, CA). Cells were then allowed to incubate for 48 h at 37°C before cell viability was determined using the CellTiter 96^® AQ_ueous One Solution Reagent assay (Promega, Madison, WI) per the manufacturer’s protocol.
Cells were passaged using 0.25% trypsin-EDTA followed by counting (ViaStain AOPI Staining Solution,cridine orange/propidium iodide Nexcelom Bioscience, Lawrence, MA) using an automated cellometer (Cellometer K2, Nexcelom Bioscience, Lawrence, MA) and then harvested at 90% confluency.