Measuring your cells' total ATP level provides you with a piece of the story. However, without a view on the rate of production or pathway contribution, you may be missing important insight into cell proliferation, differentiation, activation and death.
The Agilent Seahorse XF Real-Time ATP rate assay allows you to measure what matters. Now you can measure the rate of ATP production from the two key energy pathways (glycolysis and mitochondrial respiration) simultaneously in live cells using label-free technology. Unlike traditional end-point total ATP assays, this real-time, dual-pathway kinetic quantification delivers a dynamic picture of cellular bioenergetics with pathway contribution indicators, delivering earlier and deeper insights over other cell analysis techniques.
Quantify ATP production rate from both glycolytic and mitochondrial pathways.
Monitor kinetic responses in ATP production rate from drug or compound dosing.
Key outputs of Agilent Seahorse XF Real-Time ATP rate assays. A: Illustrates quantitative metabolic shift from mitochondrial respiration to glycolysis, resulting in different phenotypes under different conditions. B: Shows the changes of mitoATP production rates and glycoATP production rates over a time course in one cell population.
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White Paper to learn how these measurements are obtained.
Gain a New Perspective - What is ATP and its measure to you?
Cellular energy metabolism is a fundamental driver of cell phenotype and function. This energy is generated in the form of ATP through glycolysis and/or mitochondrial respiration, and is essential for all cellular activities. With the Agilent Seahorse XF Real-Time ATP rate assay, you can measure and quantify these rates of ATP production from both pathways simultaneously in live cells. Kinetic quantification of ATP production provides a powerful new way to measure cell function.
Measure ATP Production Rate - Go beyond ATP level assays
Go beyond end-point total ATP assays and gain a new perspective on ATP dynamics
Cellular ATP levels are maintained through a highly regulated system that allows
cells to respond to changes in ATP demand via changes in ATP production rates.
This system results in the maintenance of constant cellular ATP levels under
With that being said, assays that measure total ATP levels do not provide dynamic
information about cellular activities and energy demand.
The real-time quantification of ATP production
offers a more informative approach to assess the interplay
between energy metabolism and cellular functions in response to gene
modification, compound exposure, and/or any other types of interventions.
Rate measurements provide a unique insight into cellular activities beyond end-point total ATP level assays
When A549 cells were exposed to monensin, an Na+ ionophore, ATP production rates measured by the XF Real-Time ATP rate assay were boosted by 25 % due to increased Na+ ion import and subsequent Na+/K+ ATPase activity. When A459 cells were exposed to cycloheximide, an inhibitor of peptide elongation during translation, ATP production rates were reduced ~20 % due to decreased ATP demand.
In both cases, cellular ATP levels measured by a standard luminescent total ATP assay were constant. This demonstrates that measuring the rate of ATP production is a more informative method for monitoring dynamics of cellular function and metabolism than static total cellular ATP measurements.
Get Definitive Phenotype - Quantify dual ATP pathway responses
Quantify metabolic switching and pathway liabilities with the definitive measure of cell energy metabolism
Metabolic switching reveals the ability of a cell to compensate for reduced or lost function of one metabolic pathway by using an alternative pathway to meet energy demands for cellular activities. The Seahorse XF Real-Time ATP rate assay provides a complete cell energy profile, with relatable mitochondrial and glycolytic pathway production rates, enabling researchers to quantify metabolic switching in response to modulators or uncover pathway or fuel liabilities.
Simultaneous quantification of both energy producing pathways provides relatable switching data
MCF7 cells obtain ~70 % of their ATP via mitochondrial respiration under normal culture conditions. When mitochondrial respiration is partially inhibited by modulator UK5099 at low concentration, glycoATP production rate increases to compensate. This compensation results in a constant total ATP production rate to meet the energy demand for cellular activities.
When glycoATP production is inhibited by starving cells of glucose and providing galactose, MCF7 cells switch to and elevate mitoATP production rate to compensate for the loss of glycolysis.
Rely on a Validated Methodology - Assay design and XF Report generator
The new Seahorse XF Real-Time ATP rate assay utilizes XF technology
to detect mitochondrial Oxygen Consumption Rates (OCR) and glycolytic
Extra-Cellular Acidification Rates (ECAR).
These data are then transformed to mitoATP and glycoATP production rates
using well-validated algorithms.
This assay removes contributions from non-ATP production related signals
through sequential injection of energy pathway modulators
(oligomycin and a mixture of rotenone and antimycin A).
In this way, accurate measurements are achieved for mitoATP
and glycoATP production rates, as well as the total ATP production rates.
The data analysis
Seahorse XF Real-time ATP rate assay report generator is a powerful data analysis tool, which is offered together with the assay kits. Raw data can be imported into the data analysis tool and automatically processed. Final data are organized in shareable and easy-to-understand summary reports with publishable graphs. Examples of the graphs include stacked bar chart (A), energetic map (B), XF ATP rate index (C), and kinetic trace of ATP production rates (total, glycoATP and mitoATP) (D).
Uncover the benefits you can now introduce to your research
Easy-to-run assay on Agilent Seahorse Analyzers, models XFe96, XF96, XFe24, and XFp
Integrated data processing tools for post assay analysis
Optimized single-use format for a simplified workflow and reduced assay complexity
Energy metabolism in immune cells has become the focus of intense investigation in recent years, revealing tight connection between metabolic pathways and immune responses. Agilent Seahorse XF Technology enables researchers to understand the role of energy metabolism in immune response and modulation in real-time.
Discover how the Seahorse XF Real-Time ATP rate assay can be used to study immune cell modulation and lineage commitment.
Altered cellular metabolism is a hallmark of cancer and has been a key driver in cancer research for many year. Agilent Seahorse XF Technology measures changes in both glycolytic and mitochondrial activity in real time, enabling researchers to understand how and why metabolic reprogramming occurs in cancer cells.
Discover how the Seahorse XF Real-Time ATP rate assay can be used to profile metabolic phenotype for cancer cells and link cancer behavior to energy metabolism.
The next generation of immune cell therapies require a new generation of analysis tools.
A safe, potent, and persistent immune cell product depends on developers mustering the full repertoire of immune cell function; activation, proliferation, cell fate, cytotoxic killing, immunomodulation, and memory. All of which must be accomplished in the ever-changing, suppressive, and toxic tumor microenvironment.