RealTime-Glo™ MT Cell Viability Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 1 × RealTime-Glo MT Cell Viability Assay reagent to the culture	Luminescence was read on a Tecan Infinite M200 plate reader at 3, 6, 12, 22, 31, and 47 h.

Protocol tips
Add 1 × RealTime-Glo MT Cell Viability Assay reagent to the culture
Downstream tips
Luminescence was read on a Tecan Infinite M200 plate reader at 3, 6, 12, 22, 31, and 47 h.

Publication protocol

The library was screened with K562 cells. This cell line represents a commonly used cancer cell type that is grown in suspension. To the predispensed compounds, 5 μL of RPMI media was added. K562 cells (1,500/well) were plated in 20-μL RPMI media containing 1 × RealTime-Glo MT Cell Viability Assay reagents. Luminescence was read on a Tecan Infinite M200 plate reader at 3, 6, 12, 22, 31, and 47 h. The protocol for the screen performed using the real-time cell viability assay is described in Table 1. Two separate sets of the library were used for two independent screens to compare the reproducibility of the RealTime-Glo MT Cell Viability Assay. All the reagents were dispensed using Multidrop Combi (Thermo Scientific). The robustness of the assay was determined by calculating the signal-to-background (S/B) (average signal from vehicle control cells/average signal from 100-μM benzethonium chloride-treated cells), signal-to-noise (S/N) ([average signal from vehicle control cells/average signal from 100-μM benzethonium chloride-treated cells]/standard deviation from 100-μM benzethonium chloride-treated cells), and Z′ value.9 Z′ was calculated using the values from the vehicle control cells (signal) and 100-μM benzethonium chloride-treated cells (inhibited).

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Manufacturer protocol

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