An Adenosine Triphosphate (ATP) assay is a laboratory method used to measure the amount of ATP, the primary energy molecule, within biological samples. The concentration of ATP is a strong indicator of metabolic activity, and because living cells actively produce it while dead cells do not, these assays are a widely accepted way to determine the number of viable cells. These measurement techniques are developed by various life science companies, with Promega Corporation being a significant contributor to these technologies that are now commonplace in research and industrial settings.
The Bioluminescent Principle of Promega Assays
At the heart of Promega’s ATP detection systems is the bioluminescent reaction of firefly luciferase. This process involves an enzyme, luciferase, and a substrate, D-luciferin. The reaction requires ATP to proceed, making it the limiting component. When ATP is present, it provides the energy for luciferase to convert D-luciferin into oxyluciferin, releasing energy as light (around 550–570nm).
The quantity of light produced in this reaction is directly proportional to the amount of ATP available. If there is a large amount of ATP, the reaction proceeds rapidly, generating a strong light signal. Conversely, a small amount of ATP results in a much weaker signal. This relationship allows for precise quantification.
To measure this light output, scientists use a luminometer, which detects and quantifies the photons emitted during the luciferase reaction. By measuring the light intensity, researchers can deduce the concentration of ATP in their original sample. The stability of the light signal has been enhanced through protein engineering, allowing for a consistent “glow” that lasts for hours, a significant improvement over early assays that produced only a brief flash of light.
Promega’s ATP Assay Portfolio
Promega offers a diverse range of ATP assay kits categorized into two main types: lysis-based endpoint assays and non-lytic real-time assays. The lysis-based method is the most common, providing a terminal measurement of ATP within a cell population at a single point in time. These assays work by lysing cell membranes to release the entire intracellular ATP pool.
A prominent example of a lysis-based kit is the CellTiter-Glo® Luminescent Cell Viability Assay. This system uses a single reagent containing a detergent to break open cells and release ATP, plus the luciferase and luciferin for the reaction. The formulation produces a stable, long-lasting luminescent signal, simplifying the workflow to an “add-mix-measure” process.
In contrast, non-lytic assays are designed to measure cell viability continuously without killing the cells. This allows researchers to monitor the health of the same cell population over extended periods. The RealTime-Glo™ MT Cell Viability Assay exemplifies this approach, using a prosubstrate that is taken up by living cells and converted into the active luciferin substrate.
This active substrate is then released into the culture medium where it reacts with a specially engineered luciferase, also present in the medium, to produce light. Because this process relies on the metabolic activity of living cells to generate the substrate, the light signal provides a real-time indicator of cell health.
Promega has developed specialized versions of these assays to accommodate various research questions. For instance, the BacTiter-Glo™ Microbial Cell Viability Assay is optimized for lysing and measuring ATP in bacterial cells. The CellTiter-Glo® 3D Assay is formulated with enhanced lytic capacity to penetrate and measure viability in three-dimensional cell culture models.
Key Applications in Research
ATP assays are used in many areas of biological research to assess cellular health and growth. One widespread application is in cytotoxicity screening, where researchers expose cells to compounds, such as potential drug candidates, to determine toxicity. A decrease in the luminescent signal indicates the compound has killed cells or impaired their metabolic function.
These assays are also fundamental to studies of cell proliferation. By taking measurements at different time points, scientists can track the increase in ATP concentration as a cell population grows. This application is valuable for understanding the effects of growth factors, nutrients, or inhibitory substances on cell division rates.
The simple nature of these assays makes them well-suited for high-throughput screening (HTS) in academic and pharmaceutical settings. The “add-mix-measure” protocol minimizes hands-on time and can be easily automated, allowing for the rapid testing of thousands of compounds. The high sensitivity of bioluminescent detection means that assays can be performed in high-density microplates with very small cell numbers, making it a cost-effective and efficient method for large-scale drug discovery projects.
Assay Selection and Practical Considerations
Choosing the appropriate ATP assay requires consideration of the experimental goals. A primary decision is whether the experiment requires a single endpoint measurement or continuous monitoring. For determining cell viability at a fixed time after treatment, a lysis-based assay like CellTiter-Glo® is a direct and sensitive option. For tracking dynamic changes in cell health, a non-lytic, real-time assay like RealTime-Glo™ is more suitable.
The cell type being studied is another important factor. While many assays work with standard mammalian cell lines, specialized kits are available for challenging sample types. Bacteria or cells in 3D cultures may require reagents with stronger lytic capabilities to ensure complete ATP release. Assays with stable “glow” signals are preferable for HTS as they provide a flexible window for reading plates.
To ensure accurate and reproducible results, several practical considerations must be addressed. It is standard practice to include proper controls, such as wells with no cells (blanks) to measure background luminescence and wells with untreated cells to establish a baseline for normal viability. For absolute quantification of ATP, a standard curve using known concentrations of ATP should be prepared. Researchers must also be vigilant about potential sources of ATP contamination from equipment or reagents.