A Helpful Introduction to the ATP Bioluminescence Assay

Cell viability assays are widely employed by researchers to assess the overall health and functionality of cells. These assays play a crucial role in optimizing experimental conditions, gaining insights into cellular metabolism, and evaluating the impact of specific treatments. In this article, we will focus on the ATP bioluminescence assay—a technique that leverages the interaction between ATP, luciferin, and luciferase to estimate the number of viable cells.

What is the ATP Bioluminescence Assay?

The ATP bioluminescence assay relies on the principle that adenosine triphosphate (ATP) is present only in metabolically active, living cells, and that intracellular ATP levels decline rapidly once a cell dies. By detecting ATP-dependent bioluminescence, researchers can estimate the number of viable cells within a given sample.

In living cells containing ATP, the luciferin–luciferase reaction generates a measurable burst of light. In contrast, dead cells lack ATP and therefore produce no bioluminescent signal.

When performed under controlled conditions, the intensity of the emitted light is directly proportional to the ATP concentration, which serves as an indicator of the number of viable cells in the sample (Brovko, 2010).

How Does the ATP Bioluminescence Assay Work?

The ATP bioluminescence assay operates by introducing the enzyme luciferase and its substrate luciferin to a cell sample. Because the luciferase reaction is ATP-dependent, only metabolically active (living) cells generate a bioluminescent signal.

In contrast, dead cells exhibit disrupted metabolism. While cellular ATP production ceases, ATP-degrading enzymes (ATPases) remain active for a short time, leading to a rapid depletion of intracellular ATP. As a result, no light is produced from these cells.

To fully grasp the basis of this assay, it’s important to understand the luciferase reaction itself.

The Luciferase Reaction

This reaction proceeds in two main steps:

1. Adenylation of luciferin –  ATP is consumed to convert luciferin into luciferyl adenylate.

2. Oxidative decarboxylation – The luciferyl adenylate undergoes oxidation, producing oxyluciferin in an excited state. As oxyluciferin returns to its ground state, a photon (light) is emitted (Belanger & University of Rochester Introductory Biochemistry, 2018).

Figure 2. Basic firefly luciferase reaction with ATP and D-luciferin. Light emission occurs when excited oxyluciferin transitions back to the ground state.

This reaction demonstrates that ATP is an essential component for bioluminescence. Since ATP is exclusively present in living cells, researchers can harness this biochemical pathway to visually and quantitatively distinguish between viable and non-viable cells in a sample.

Applications of the ATP Bioluminescence Assay

The ATP bioluminescence assay is a versatile tool used across a wide range of research and industrial fields (Riss et al., 2016; Brovko, 2010), including:

• Sanitation assessment
• Food safety and food processing
• Biocide dosing optimization
• Fermentation management
• Cell culture optimization
• Experimental condition refinement
• Evaluating treatment efficacy
• Cytotoxicity studies
• Receptor binding measurements
• Signal transduction analysis
• Pathogenicity research
• Oncology studies
• Drug discovery and development

Substrates Used in the ATP Bioluminescence Assay

Because the assay is based on the luciferase reaction, firefly luciferin is the primary substrate.

However, other bioluminescent viability assays employ different systems. Renilla and Gaussia luciferase assays use the substrate coelenterazine. A notable advantage of the Gaussia system is that the enzyme is secreted from the cell, enabling easier in vivo detection and facilitating high-throughput screening. That said, Renilla and Gaussia-based systems have a key limitation: their light emission (~480 nm) is strongly absorbed and scattered by mammalian tissues, which reduces sensitivity for deep-tissue in vivo imaging (Brovko, 2010).

Challenges with ATP Bioluminescence Substrates

While firefly luciferin readily crosses the eukaryotic cell membrane, it faces permeability challenges with bacterial cells due to its negative charge in solution. Several strategies can help address this:

1. pH Adjustment

• Lowering the pH to a slightly acidic level allows protonation of luciferin’s carboxylic group, improving permeability.
• Caution: avoid pH values below 5.0, as this will inactivate luciferase.

2. Polymyxin B Treatment

• Polymyxin B creates pores in E. coli membranes, allowing luciferin entry.
• Use with care, as high concentrations can be lethal to cells.

3. Caged Luciferins

• These modified, neutral substrates more easily penetrate the bacterial cell wall.
• Once inside, they can be converted to active luciferin via enzymatic action or photoactivation (Brovko, 2010).

References

AIB Staff. (2013, October 11). A Q&A on ATP Bioluminescence assay. Retrieved March 22, 2021, from https://www.qualityassurancemag.com/article/aib1013-atp-bioluminescence-assay/

Belanger, S., & University of Rochester Introductory Biochemistry (Producers). (2018, May 15). Luciferase [Video file]. Retrieved March 22, 2021, from https://www.youtube.com/watch?v=odN92KbH8Bo

Brovko, L. (2010). Bioluminescence and fluorescence for in vivo imaging: In vivo optical imaging (Vol. TT91). Bellingham, WA, Washington: SPIE Press.

Riss, T., Moravec, R., Niles, A., Duellman, S., Benink, H., Worzella, T., & Minor, L. (2016). Cell Viability Assays. The Assay Guidance Manual.

Aladdin: https://www.aladdinsci.com/