High-throughput 1536-well Cell Viability Assay

A 1536-well 2D cell viability assay was optimized and implemented, which determines the number of viable cells based on the amount of ATP present using commercially available luminescence detection reagent CellTiter-Glo (Part# G7573, Promega, Madison, WI) as previously described.^{22 (link)}Prior to plating, cells were grown to 80% confluence in RPMI 1640 complete growth media. After washing once with PBS, cells were detached by TrypLE (Part# 12604021, Life Technologies, Carlsbad, CA) and centrifuged at 300 × g for 5 min. Cells were suspended and filtered through cell strainer. 200 cells in 5 μL culture media were seeded in 1536-well plates (Part# 789173-F, Greiner, Monroe, NC). After incubation of the assay plates overnight (~14 hours), cells were treated with compounds and vehicle (10 nL, 0.02% DMSO). Cell viability was assessed after 72-hour incubation using Cell-Titer Glo reagent according to manufacturer’s instruction. The ViewLux microplate reader (Perkin Elmer, Waltham, MA) was used to quantitate luminescence signal. IC₅₀ values of 5 pharmacological control compounds (Doxorubicin, Gemcitabine, SN-38, 5-Fluorouracil, and Oxaliplatin, all purchased from Sigma) were determined by fitting the concentration response curve data (CRC) with a four-parameter variable slope method in GraphPad Prism (GraphPad software, San Diego, CA).