Your interactive guide
to choosing the right assays
Select the assays that will probe the correct pathways and deliver deep insights
Agilent Seahorse XF assay kits work with your Seahorse XF analyzer to elucidate the role of energy metabolism in cellular processes. These insights can be used to identify potential druggable gene and protein targets and to validate potential metabolic modulators in early drug discovery research.
Here, you’ll find a guided assay journey strategy that starts with streamlined cell metabolism assays, then digs deeper with pathway-specific assays.
Screen for metabolic phenotype and identify the pathway involved
The XF Real-Time ATP Rate Assay kit provides a streamlined protocol and an ideal entry point for cell metabolism analysis.
In addition to whole-cell metabolic changes, you will get a dynamic picture of mitochondrial and glycolytic bioenergetic flux. Simultaneous assessment of both pathways using a common unit of measurement enables quantitative analysis of metabolic switching.
Seahorse XF Real-Time ATP Rate Assay kit
This quantitative method simultaneously measures the production rates of adenosine triphosphate (ATP) from mitochondrial respiration and glycolysis in live cells.
Screen and assess metabolic effects
Visualize the effects of drug candidates on live-cell metabolic function (quiescent to energetic) as well as metabolic shift between pathways (aerobic versus glycolytic).
ATP production rate is a sensitive functional measurement of the two major cell metabolism mechanisms: glycolysis and mitochondrial respiration.
Pathway-specific ATP production rates deliver simplified outputs and actionable results compared to extracellular acidification rate (driven by both glycolysis and mitochondrial respiration).
Quantification of pathway-specific contributions in pmol ATP/min allows for ratiometric analysis and fast identification of metabolic switching.
Pathway-specific heat map and dose response views
Mitochondrial respiration
Glycolysis
Measure dose-dependent mitochondrial effects.
Rapidly identify mitochondrial changes.
Quickly identify glycolytic changes.
Evaluate dose-dependent glycolytic effects.
With your ATP rate assay results, you can now probe deeper using
pathway-specific assays.
Mitochondrial respiration
Glycolysis
Functional analysis of glycolysis
If a glycolytic pathway is determined, you’ll need to measure the effects of metabolic modulators on glycolytic rates, and detect transient and rapid metabolicswitches.
Seahorse XF Glycolytic Rate Assay kit
Accurately measure glycolysis in live cells by quantifying proton efflux rate (PER) specific to glycolysis.
Live-cell kinetic glycolytic rate assay profile
These data represent uncorrected ECAR (driven by both glycolysis and mitochondrial respiration) before glycolytic PER quantification.
Glycolytic protein efflux rate is a quantitative measure of cellular glycolysis.
Multiparametric analysis
Quantitative basal glycoPER reflects normal (physiological) glycolysis.
Cells can upregulate glycolysis when mitochondrial respiration is inhibited. Compensatory glycolysis reflects cellular ability to manage energy demands under stress.
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Functional analysis of mitochondrial respiration
Once you determine that aerobic respiration is involved, you’ll want to gain a comprehensive understanding of mitochondrial function under basal and stressed conditions.
Common drug discovery target parameters, like spare respiratory capacity and maximal respiration, are calculated automatically.
Seahorse XF Cell Mito Stress Test Assay kit
This assay is a well-recognized method for evaluating mitochondrial function by measuring multiple key parameters.
Live-cell kinetic mito stress test profile
This kinetic graph shows the real-time oxygen consumption rate through a sequence of inhibitor and uncoupler injections.
Major parameters that characterize mitochondrial function are calculated automatically.
Multiparametric analysis
Next step: Mechanisms of action
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Targeted assessment of mitochondrial mechanisms
Substrate utilization and dependency
Understanding fuel utilization and dependency allows you to characterize and modulate specific cellular functions and/or phenotypes.
Seahorse XF Substrate Oxidation Stress Test Assay kits
With these optimized kits, you can dive deeper to study the impact of three primary substrates on mitochondrial and cellular function.
Using pathway-specific inhibitors, XF Substrate Oxidation Stress Test assays demonstrate fuel-specific dependencies.
Seahorse XF Palmitate Oxidation Stress Test kit
Investigate how interventions specifically affect palmitate oxidation when starved of other fuels.
By forcing cells to use the long-chain fatty acid palmitate, pathway-specific effects can be captured.
Mitochondrial complexes
Selectively targeting the cellular plasma membrane while leaving the mitochondrial membrane intact can help you characterize key components of mitochondrial function.
Seahorse XF Plasma Membrane Permeabilizer
Our proprietary reagent permeabilizes intact cells in culture, so you can perform mitochondria-type assays without isolating mitochondria.
Permeabilize the plasma membrane while leaving the mitochondria intact. This process allows you to determine which mitochondrial complexes are affected.
Interested in additional assays for cell therapy development or drug safety assessment?
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Application-specific tools for cell therapy development and preclinical safetystudies
To make informed decisions, you need to assess key attributes pertaining to the safety and efficacy of potentialtreatments.
T cell metabolic profiling
When developing therapies, you need to determine whether your T cells can support a long-term immune response. You also need to know if your cells are metabolically poised to survive in the tumor microenvironment.
Seahorse XF T Cell Metabolic Profiling kit
Generate robust bioenergetic parameters linked to critical attributes for antitumor potency: T cell persistence and T cell metabolic fitness.
Seahorse XF T Cell Metabolic Profiling Assays provide a comprehensive view of cellular bioenergetics, including respiratory capacity and metabolic profile (glycolytic vs respiration rates).
Mitochondrial toxicity
As many as 25% of drug failures can be attributed to safety—and mitochondrial disfunction is the leading cellular mechanism. That’s why it’s critical to identify mitochondrial toxicity during early drug discovery.
Seahorse XF Mito Tox Assay kit
Identify drug-induced mitochondrial toxicity through functional measurements of mitochondrial oxygen consumption rates.
The XF Mito Tox Assay provides easy identification of mitochondrial toxicants and distinguishes between modes of action (inhibitors vs uncouplers).
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Deepen your investigation into the role of energy metabolism in cellular processes
Agilent Seahorse XF assay kits
Now that you’ve learned about Seahorse XF assays, visit our product pages for a more in-depth look, and to purchase cell analysis assays and supplies.
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Agilent Seahorse XF Pro analyzer
Developed for pharma, the Seahorse XF Pro analyzer features advanced experimental design and analysis tools, so you can optimize your workflow from start to finish.
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Discover how XF Pro technology can advance your drugdiscovery studies
Detect rapid, real-time changes in cellular bioenergetics with a platform optimized for pharma-oriented workflow solutions, whether your drug discovery strategy is small molecule, biologic, or cell therapy.
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Basal respiration is the starting oxygen consumptionrate.
ATP production-coupled respiration is the portion of basal respiration used to drive ATP production.
Proton leak can be a sign of mitochondrial damage. It can also be used as a mechanism to regulate mitochondrial ATP production.
Maximal respiration reflects full mitochondrial capability.
Spare respiratory capacity is a metric used to evaluate the cells’ ability to meet an energetic challenge.
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