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The Mitochondrial Research Core has standard assays that we provide to assess mitochondrial function. We can also consult on various methods and approaches to assess mitochondrial changes in different physiologic contexts.  

Electron Transport Chain (ETC) Assays

We provide access to a Seahorse XF96 Analyzer, a specialized instrument used to measure the real-time metabolic activity of live cells by quantifying their oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), allowing researchers to study cellular bioenergetics and assess the effects of drugs or treatments on cellular metabolism without the need for labels or dyes. 

Users are responsible for supplying consumables for the instrumentation. 

We perform 8 assays to assess the activity of individual ETC complexes. A protein assay is performed for normalization followed by the following assays:

• NCR   NADH-cyt c reductase (rotenone sensitive)
• SCR   Succinate-cytochrome c reductase
• CII   Succinate-Q reductase +/- duroquinone (TTFA sensitive)
• SDH   Succinate Deydrogenase
• CIV   Cytochrome C Oxidase
• CIII  Decylubiquinol-cytochrome c reductase
• CS    Citrate Synthase
• LDH   Lactate Dehydrogenase

*Note: It is generally beneficial to examine the collective changes in ETC activities as a composite with these assays. If this is needed, we can arrange a special project for a subset of assays after consultation.

Oroboros O2K

High-resolution respirometry (HRR) is the state-of-the-art approach in mitochondrial research to measure cellular respiration in various types of mitochondrial preparations from tissues and living cells. Intact cells are assessed for basal respiratory function. Subsequent permeabilization allows for the controlled introduction of ETC substrates. In parallel protocols, we add pyruvate, malate, and glutamate as complex I substrates, succinate for complex II, duroquinol (DHQ) for complex III, TMPD with Ascorbate for complex IV, and palmitoylcarnitine  to assess fatty acid oxidation (FAO). In a predetermined sequence, these substrates and complex inhibitors (i.e rotenone for complex I and antimycin A for complex III) are added to distinguish individual ETC complex activities in the cell preparations.

The Oroboros O2k assays are currently offered as a fee-for-service. The cost per sample includes parallel runs in two chambers with two different protocols that examine different ETC activities in the cells/sample. From these assays, rates of Complex I, II, III, IV and FAO are assessed at maximal rates (uncoupled) and linked to ATP synthesis.

*Note: The O2K is capable of performing tailored experiments to examine different substrates. If this is needed, we can arrange a special project after consultation.

Mass Photometry 

Mass Photometry (MP) is used to rapidly determine mass, oligomeric state, and heterogeneity of a wide range of macromolecules and their complexes in various conditions.

Supporting publications: 

Organization Affiliations: Center for Mitochondrial Research & Therapeutics