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Functional Mitochondrial Toxicity Assay (using Seahorse XFe96 flux analyzer)

Understand cellular bioenergetics and specific mechanisms of mitochondrial toxicity using Cyprotex’s functional mitochondrial toxicity assay.

Cyprotex’s functional mitochondrial toxicity service is a cell based assay which uses the Seahorse XFe extracellular flux analyzer is in Cyprotex's portfolio of in vitro toxicology services for measuring potential mitochondrial toxicity. Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Mitochondrial toxicity and its measurement in vitro using the Seahorse Flux Analyzer

  • Impairment of mitochondrial function is increasingly implicated in the etiology of drug-induced toxicity.1
  • The Seahorse XFe96 extracellular flux analyzer is used to detect, in real time, effects of compounds on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in order to assess mitochondrial function and cellular metabolism.
  • The assay uses the mitochondrial stress test  to gain an insight into cellular bioenergetics and the mechanism of mitochondrial toxicity.2
  • In the stress test, cells are exposed sequentially to oligomycin (ATP synthase inhibitor), FCCP (protonophoric uncoupler), and rotenone and antimycin A (electron transport inhibitors). This provides information on basal respiration, proton leak, maximum respiration rate, and non-mitochondrial respiration.
  • As well as mitochondrial toxicity, the Seahorse XFe flux analyzer can be used for other applications where a shift between mitochondrial respiration and glycolysis is observed under certain pathological states (e.g., obesity, diabetes, cancer, cardiovascular disease and neurodegenerative function).
Drug-induced mitochondrial toxicity is rapidly gaining recognition within the pharmaceutical industry as a contributor to compound attrition and post-market drug withdrawals.

3 Nadanaciva S and Will Y (2011) Current Pharmaceutical Design 17; 2100-2112


Functional mitochondrial toxicity assessment protocol

Media Assessed Unbuffered DMEM containing 10 mM glucose, 1 mM pyruvate and 2 mM glutamine
Cell Types Available* H9c2, Huh7, HepG2, MCF-7, cropreserved human hepatocytes, cryopreserved rat hepatocytes (other custom cell lines available on request)
Test Article Concentration 7 point dose response
Quality Controls Vehicle control
Rotenone (positive control)
Test Article Requirements 50 µL of 50 mM DMSO solution or equivalent amount of solid compound.
Analysis Method Use of solid state fluorescent sensors to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Measured using the XFe96 flux analyzer (Seahorse Biosciences Inc)
Data Delivery Summary report
AC50 for OCR, reserve capacity and ECAR
Minimum effective concentration (MEC) for OCR, reserve capacity and ECAR

Related Services
HCS based mitochondrial toxicity assessment
Glucose/galactose mitochondrial toxicity assessment


Data for functional mitochondrial assay

Known mitochondrial toxicants and non-toxicants were screened in the Seahorse assay.




Figure 1
Effect of rotenone on A) oxygen consumption rate and B) extracellular acidification on H9c2 cells.

The addition of rotenone following the 4 basal reading results in a dose dependent decrease in (A) OCR, and compensatory increase in (B) ECAR. Following the addition of oligomycin, there is a decrease in OCR as expected, demonstrating no increase in proton leak. In the presence of FCCP, the OCR increases, and is a measure of the reserve capacity of the cells. There is a dose dependendent decrease in the reserve capacity of the cells exposed to rotenone, as expected since it is a known inhibitor of complex I of the electron transport chain.
  Oxygen Consumption Rate (OCR)Reserve CapacityExtracellular Acidification Rate (ECAR)
CompoundMechanismMEC (µM)AC50 (µM)MEC (µM)AC50 (µM)MEC (µM)AC50 (µM)
Rotenone Complex I inhibitor 0.008 0.017↓ 0.01 0.021↓ 0.01 0.016↑
2-Thenoyltrifluoroacetone Complex II inhibitor 6.5 46.4↓ 5 17.5↓ 48 35.8↑
Myxothiazol Complex III inhibitor 0.1 0.18↓ 3 1.8↓ 3 1.0↑
Antimycin A Complex III inhibitor 0.01 0.012↓ 0.01 0.008↓ 0.01 0.010↑
Oligomycin Complex V inhibitor (ATP-synthase inhibitor) 0.1 0.11↓ NR NR 0.3 0.12↑
Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) Uncoupler 0.1 0.25↑ 10 1.7↓ 0.1 0.10↑
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) Uncoupler 0.1 0.14↑ 1 1.0↓ 0.1 0.044↑
2,4-Dinitrophenol Uncoupler 3 4.9↑ NR NR 3 1.4↑
Etomoxir β-oxidation inhibitor 7 94.9↓ NR 67.9↓ 7 NR
UK-5099 Pyruvate transport inhibitor 19.3 92.1↓ 0.1 2.3↓ 0.09 NR
2-Deoxyglucose Glycolysis inhibitor NR NR NR NR NR NR
Methapyrilene No evidence NR NR NR NR NR NR
Physostigmine No evidence NR NR NR NR NR 4.5↑
Betaine No evidence NR NR NR NR NR NR
Streptomycin No evidence NR NR NR NR NR NR
NR = no response

Table 1

Effect of test compounds on OCR, Reserve Capacity and ECAR.


1 Dykens JA and Will Y (2007) The significance of mitochondrial toxicity testing in drug development. Drug Discovery Today 12; 777-785
2 Brand MD and Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435; 297–312
3 Nadanaciva S and Will Y (2011) New insights in drug-induced mitochondrial toxicity. Current Pharmaceutical Design 17; 2100-2112

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