ATP Assays

What is an ATP Assay?

ATP is the energy source of all living cells and is involved in many vital biochemical reactions. When cells die, they stop synthesizing ATP and the existing ATP pool is quickly degraded. Therefore, ATP is widely accepted as a marker of viable cells. Higher ATP concentration indicates higher number of living cells.

ATP assays are procedures that can measure cell viability based on detection of ATP. All living cells, including bacteria, can be detected with ATP assays. Several detection methods can be used, such as colorimetric, fluorescent and bioluminescent. Most researchers choose bioluminescent ATP assays to measure cell viability due to higher sensitivity, simple and homogeneous protocol, and fast results.

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How do Bioluminescent ATP Assays Work?


Bioluminescent ATP assays take advantage of the firefly luciferase enzymatic reaction, which uses ATP from viable cells to generate photons of light. Viable cells are lysed to release the ATP for detection, and reagents containing firefly luciferase enzyme and substrate are added to catalyze a two-step reaction.

The first reaction step is luciferin activation with ATP to give a luciferyl-adenylate and pyrophosphate. In the second step, the luciferyl-adenylate reacts with molecular oxygen to yield oxyluciferin in an electronically excited state and CO2. The excited-state oxyluciferin then returns to the ground state with the release of green to yellow luminescent light (550–570nm). The intensity of the luminescent signal is detected using a luminometer.

When ATP is the limiting component in the luciferase reaction, the luminescence is proportional to the ATP concentration. Higher luminescent signal indicates higher ATP levels.

Linear Correlation of Luminescence and ATP Concentration

Data generated using the Water-Glo™ System.

In early ATP assays, the luciferase reaction released a brief “flash” of light that lasted only seconds. Detection of such a short signal required a luminometer with automatic dispensing capabilities, either a single-tube or multiwell plate reader.

To improve the experimental workflow and reduce variability of assay results, Promega scientists derived a stable form of recombinant luciferase (Ultra-Glo™ rLuciferase) that is not affected by strong detergents used for lysing cells and can co-exist with ATPase inhibitors, which stabilize ATP once released from lysed cells. The enhanced stability and flexibility of Ultra-Glo™ rLuciferase enabled development of ATP assays with a luminescent signal that “glows” for up to hours compared to the historical “flash” assays that lasts only seconds.

Glow-type ATP assays offer researchers a more flexible workflow, enabling a significantly larger number of samples to be assessed in an experiment, and do not require automatic dispensers for any protocol steps.

How to Choose an ATP Assay

The first step in choosing an ATP assay is understanding what you are using it for. ATP has many functions in the cell and cellular environment. Therefore, ATP assays have a wide range of applications. In research labs, they are commonly used in cell culture experiments to estimate the number of viable cells. This information can answer questions about cell growth, or assess toxicity of drugs or viruses.

Extracellular ATP released from dying cells can act as a damage associated molecular pattern (DAMP), and specialized ATP assays can be used for applications such as measuring immunogenic cell death. Outside of basic biomedical research, ATP assays are also used to determine the presence of bacterial contamination in water samples, food products and cosmetics.

Besides understanding the intended use, a variety of factors will influence which ATP assay to choose. These include the nature of the sample, the number of samples being tested, the required sensitivity, and availability of automated liquid handlers and detection instruments. In addition, researchers performing high-throughput screening or using 3D cell culture will want to choose an assay that has been tested and optimized for these applications.

Choosing an ATP assay can be challenging due to the large number of products available. To support the broad applications of ATP assays, we have developed a portfolio of luminescent ATP assays with distinct formulations and recommended protocols optimized for each application. See the table below to find an assay that would work best for your application.

Need help choosing the right assay for your needs? Our Technical Support Team would be happy to guide you!

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Product Sample Type Description Lytic Capacity Signal
Ready-to-use Single Reagent HTS Capability
CellTiter-Glo® 2.0 Cell Viability Assay Monolayer/suspension cell culture The most convenient ATP assay for high-throughput applications or daily use. Provided as a single reagent in liquid format, optimized for room temperature storage stability enabling easy workflow implementation. Strong >3 hours
CellTiter-Glo® Luminescent Cell Viability Assay Monolayer/suspension cell culture The original gold standard assay for HTS, lot release testing and general research applications. Provided as lyophilized substrate and buffer components, prepared into a detection reagent just prior to use. Strong >5 hours
CellTiter-Glo® 3D Cell Viability Assay 3D cell culture Provided as a single reagent with high detergent concentration, specifically optimized for effective cell membrane lysis in 3D microtissues. Very strong >3 hours
RealTime-Glo™ Extracellular ATP Assay Monolayer/suspension, 3D cell cultures Live-cell kinetic assay to detect ATP released from dying cells. Non-lytic assay Continuous luminescent monitoring for up to 24 hours
Viral ToxGlo™ Assay Viral-infected monolayer/suspension cell culture Protocol optimized to determine viral-induced cytopathic effect on host cells in vitro. Strong >5 hours
BacTiter-Glo™ Microbial Cell Viability Assay Bacterial cells in culture medium Measures the number of viable bacterial cells. Designed for difficult-to-lyse bacterial samples. Can be used for single sample analysis or high-throughput applications. Very strong >30 minutes
Water-Glo™ Microbial Water Testing Kit Water samples Measures the amount of biomass in complex aqueous samples, such as wastewater, seawater and freshwater. Very strong (separate lysis & detection steps) 3 minutes
ENLITEN® ATP Assay System Foodstuffs, beverages, wood pulp, cosmetics, etc. Using flash chemistry, optimize your own method to detect ATP in cells, bacteria, yeasts, fungi and other microorganisms in various sample types. Lysis reagent not included Seconds

How Are ATP Assays Used?

High-Throughput Drug Screening

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Determine Viability of 3D Cell Cultures

Learn how the original CellTiter-Glo® Luminescent Cell Viability Assay was reformulated for use with 3D cell cultures.
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Determine Viral-Induced Cytotoxicity

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