GlowQuant Assay for Cell Viability Determination

The Invitrogen GlowQuant ATP Cell Viability Assay Kit is a ready-to-use, bioluminescence-based solution designed to measure viable cells through ATP detection and quantification. The assay uses firefly luciferase to catalyze the oxidation of luciferin in the presence of ATP and oxygen, which produces a bright bioluminescent signal that is proportional to the concentration of ATP and therefore number of viable cells.

The GlowQuant assay is a firefly luciferase–based bioluminescence assay that quantifies cellular ATP used to assess cell health. Because intracellular ATP levels correlate with metabolically active, viable cells, this assay can be used to evaluate cell viability, proliferation, and cytotoxic effects. When cells lose membrane integrity, ATP production stops and endogenous ATPases rapidly deplete cytoplasmic ATP. In the presence of ATP, luciferase catalyzes a reaction that generates photons (Figure 1), and the measured light output is proportional to the ATP concentration. [1]

Figure 1. Simplified GlowQuant ATP Reaction scheme. In GlowQuant detection assays, the reagent first lyses cells to release intracellular ATP, while ATPase inhibitors stabilize the ATP after lysis. The assay then uses luciferase to catalyze the reaction of ATP with luciferin and oxygen, producing light proportional to the amount of ATP present (and therefore to the number of viable cells).

The GlowQuant assay is highly sensitive with the ability to detect low cell numbers (~50 cells/well in a 96-well plate) with a fast simple protocol, allowing for results in as little as 10 minutes (Figure 2). It requires minimal hands-on time, and provides a strong, linear bioluminescent signal for >2 hours for flexible plate reading with excellent S/B ratio, creating a larger signal window with the ability to detect smaller, more meaningful changes. Using Jurkat cells, the GlowQuant assay exhibits strong linearity and signal from 0 to 5 hours, demonstrating assay robustness for flexible plate reading (Figure 3).

Figure 2. Linearity of detection after incubation. Jurkat cells were seeded into a 96-well plate (Cat. No. 165306) in 2 fold serial dilutions and incubated overnight at 37°C. Plates were then equilibrated to room temperature, and ATP levels were measured using the GlowQuant assay in triplicate. Bioluminescence was read on a Varioskan LUX Multimode Microplate Reader. The GlowQuant assay exhibited low background and excellent linearity (R² > 0.999). The inset graph on the right demonstrates the linearity at low cell concentrations, where the GlowQuant assay can detect as low as 50 cells per 96-well.

Figure 3. Background signal intensity. Bioluminescence was read on a Varioskan LUX Multimode Microplate Reader in triplicate, where the blank wells contained solely GlowQuant reagent and other commercially availiable assays. The GlowQuant assay exhibited extremely low background and signal decay at both 0 and 5 hours.

The GlowQuant assay is a rapid, easy-to-use kit with a one-step add-and-read format. Simply add an equal volume of the reagent to the plated cells (e.g., 100 µL to a 96-well plate or 25 µL to a 384-well plate), mix the wells for 1 minute on an orbital shaker, and then incubate at room temperature for 10-20 minutes (Figure 4). Measure the luminescence with a luminometer or microplate reader, where the signal is proportional to the concentration of ATP in the cells. Damaged or non-viable cells will innately have lower ATP activity and generate less signal.

Figure 4. How the GlowQuant Assay works.

1. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK144065/