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Short answer
A Seahorse assay is a multiwell-plate respirometry assay that measures the oxygen consumption rate (OCR) of living cells. In the common Mito Stress Test format, sequential injections of oligomycin, FCCP and rotenone with antimycin A split OCR into phases, and the phase-specific values are converted into basal respiration, ATP-linked respiration, proton leak, maximal respiration and spare respiratory capacity.
Definition and scope
The method is described in the peer-reviewed literature as “a multiwell plate based respirometry assay that is widely used to quantify OCR in living cells” (OCRbayes, PLoS One 2021). It is named after the Seahorse XF instruments used to run it, but the quantity being measured is not instrument-specific: it is the rate at which cells in each well consume oxygen.
The assay is a measurement of cellular respiration, so it answers questions about mitochondrial function: how much oxygen a population of cells uses at rest, how much of that use is coupled to ATP synthesis, and how much extra respiratory activity the cells can recruit when pushed. It does not identify which mitochondrial gene, protein or pathway is responsible for a difference; that needs additional experiments.
How it works in practice
In a study running nematodes on a Seahorse XFe96 analyzer, the authors describe how “a microchamber (of 3 µL) is created at the bottom of each well in which nematode oxygen consumption is measured”, and “the decrease in oxygen is converted to a single OCR value per well” (Scientific Reports 2020). The same logic applies to cultured cells: each well yields an OCR value for each measurement period, and wells are compared across treatments.
Compounds are then added to the wells in turn, usually oligomycin, FCCP, and rotenone plus antimycin A, so that each measurement period reflects a different state of the respiratory chain. The OCR values from each phase are the raw material for every derived parameter.
The Mito Stress Test injections and the parameters they give
The table maps each injected compound to its mechanism and to what the resulting phase is used for.
| Phase or compound | Mechanism | What the phase is used for |
|---|---|---|
| Before any injection | Cells respire under assay conditions | Basal respiration |
| Oligomycin | Blocks proton translocation through ATP synthase | ATP-linked respiration and proton leak |
| FCCP | Allows protons to move into the mitochondrial matrix independently of ATP synthase | Maximal respiration and spare respiratory capacity |
| Rotenone and antimycin A | Inhibit complex I and complex III, shutting down mitochondrial respiration | Non-mitochondrial respiration |
The OCRbayes authors list the derived quantities as “basal respiration, proton leak, ATP-linked respiration, spare respiratory capacity and maximal respiration”, all calculated from the phase-specific OCR values. A study of nematodes adds non-mitochondrial respiration to the list and measures toxicity as percent OCR inhibition against a negative control.
Types and related concepts
The Mito Stress Test is the most widely described format and the one the examples above follow. The common thread in any respirometry readout is that a drug or condition is compared with a control on the same plate, and OCR is the response variable. Related plate-based readouts that answer a different question, such as cell viability, are covered separately; see the guide to the MTT assay for a viability readout that does not measure respiration directly.
Where it matters for labs and supply decisions
Respirometry results are only as good as the cell work behind them. For labs planning these experiments, the issues below are the ones most likely to change a conclusion, and they are worth settling before plates are seeded.
Interpretation checklist
- Normalize for cell number. Replicate wells differ because “cell numbers are not identical in these wells”; the OCRbayes authors report OCR value per 1000 cells. Raw OCR compared across wells with different cell counts can mislead.
- Use the same rule to pick data points per phase. The authors show that choosing different data points within a phase means “one can get different outcomes”. Fix the selection rule before looking at treatment groups.
- Watch phases near the detection limit. OCR in the oligomycin and antimycin/rotenone phases may be “possibly close to the detection limit”, which makes parameters built on them noisy.
- Do not rely on a single plate. The OCRbayes authors model between-plate variation explicitly, because plates differ; repeat experiments across plates before drawing conclusions.
Groundwork such as consistent seeding and healthy cultures matters here; the cell culture basics guide and the cell culture reference numbers cover vessel areas and seeding densities, and cell culture contamination explains a common cause of unusable wells. For background on the organelle itself, see mitochondria and their function.
Common misunderstandings
- “Seahorse” is a measurement, not a single test. The instrument reports OCR; the Mito Stress Test is one injection design layered on it.
- More oxygen use does not automatically mean healthier cells. The parameters separate ATP-linked respiration from proton leak and non-mitochondrial respiration for exactly this reason.
- A parameter is not a direct measurement. Every derived value is calculated from phase-specific OCR readings, so it inherits the noise and normalization choices of those readings.
Frequently asked questions
What does a Seahorse assay measure?
It measures the oxygen consumption rate of living cells in each well of a multiwell plate and uses injected compounds to break that rate into mitochondrial and non-mitochondrial components.
Which compounds are used in the Mito Stress Test?
Typically oligomycin, FCCP, rotenone and antimycin A, as described in the peer-reviewed literature. Oligomycin blocks ATP synthase, FCCP uncouples proton movement from ATP synthase, and rotenone with antimycin A inhibit complexes I and III.
Which parameters can be calculated?
Basal respiration, proton leak, ATP-linked respiration, maximal respiration, spare respiratory capacity and non-mitochondrial respiration.
Sources
- OCRbayes, a Bayesian hierarchical model for Seahorse OCR data, PLoS One 2021: https://pmc.ncbi.nlm.nih.gov/articles/PMC8282019/ (checked 2026-10-29).
- Oxygen consumption rate of C. elegans measured on a Seahorse XFe96 analyzer, Scientific Reports 2020: https://pmc.ncbi.nlm.nih.gov/articles/PMC7060326/ (checked 2026-10-29).
