Mitochondrial Respiratory Complex Assay
Mitochondria are the common target of drug-induced toxicity. The damage of mitochondrial function is related to the etiology of drug-induced toxicity. The permeabilization of cell membrane keeps the mitochondrial membrane intact, so that the function of mitochondria can be studied without separating mitochondria. By using complex specific substrates and inhibitors, it can identify individual complexes involved in mitochondrial toxicity.
Creative Bioarray uses permeabilized cells on the analyzer to detect mitochondrial respiratory complexes. We can provide consistent, high-quality data and flexibly adjust agreements to specific customer requirements.
Service Description
- Our extracellular flux analyzer detects the effect of compounds on oxygen consumption rate (OCR) in real time to evaluate mitochondrial function.
- Cell permeabilization that keeps the mitochondrial membrane intact allows the study of mitochondrial function without the need to isolate mitochondria.
- Single complexes (complex I, complex II, complex III, and complex IV) of the electron transport chain (etc) involved in mitochondrial toxicity can be identified using complex specific substrates and inhibitors.
- The mitochondrial respiratory complex assay can be used in conjunction with other mitochondrial assays (e.g., seahorse functional mitochondrial toxicity assay, glu/gal assay, or HCS based mitochondrial assay) to determine the possibility of mitochondrial toxicity and understanding of the mechanism.
Mitochondrial Respiratory Complex Assay Protocol
| Cell Type |
HepG2 (others available on request) |
| Analysis Platform |
Seahorse XFe96 flux analyser (Agilent Technologies) |
| Analysis Method |
Use of solid state fluorescent sensors to measure oxygen consumption rate (OCR) |
| Mechanism |
Pyruvate respiration
Succinate respiration
Ascorbate respiration |
| Test Article Requirements |
50 µL of a DMSO stock solution to achieve 100x Cmax (200x top concentration to maintain 0.5% DMSO) or equivalent amount in solid compound |
| Test Article Concentration |
7 point dose response curve with top concentration based on 100x Cmax orsolubility limit |
| Number of Replicates |
3 replicates per concentration |
| Quality Controls |
Negative control: 0.5% DMSO (vehicle)
Positive control: Assay appropriate control |
| Data Delivery |
Minimum effective concentration (MEC) and AC50 values with dose response curves for each measured parameter |
Oxygen consumption rate (OCR) of permeabilized HepG2 cells was measured in the presence of appropriate complex I substrate (pyruvate). The test compound was injected directly onto the cells and OCR was determined. After that, complex ii/iii substrate (succinate) and complex I inhibitor were injected and further measured. Finally, complex IV substrate and complex III inhibitor were added and the final OCR was determined.
The decrease of OCR after adding the test compound indicates that one of the complexes of the electron transport chain is inhibited. If this inhibition is overcome by the addition of alternative substrates, it indicates a potential inhibition site.
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All services and products are for scientific use only, not for medical use！
