Center for Metabolism
and Mitochondrial Medicine

Bioenergetics/Mitochondrial Unit

The mitochondrial/bioenergetics core provides services for the comprehensive measurement of mitochondrial function and cellular bioenergetics.  The Core, located on the 12th floor of the Biomedical Science Tower, is equipped with two Seahorse XF Bioanalyzers (96 and 24 wells), traditional respirometers, and plate readers , spectrophotometers and microscopes dedicated to the assessment of mitochondrial function by multiple techniques.  The centralization of this core allows the measurement of cellular oxygen consumption, fatty acid oxidation and glycolytic rates, individual mitochondrial enzymatic activities and expression as well as mitochondrial reactive oxygen species and apoptotic signaling.  These measurements can be made in a host of biological samples including isolated mitochondria, cultured/primary cells, tissue and blood cells.

Sruti Shiva, PhD

Director

Platform Technologies

Bioenergetic Analysis by Seahorse (oxidative phosphorylation, glycolysis, fatty acid oxidation)

Seahorse extracellular flux analysis can be used to measure rates of oxygen consumption (OCR) and extracellular acidification (ECAR) in order to probe oxidative phosphorylation, glycolysis and fatty acid oxidation in intact cells. For more information on Seahorse XF analysis see Seahorsebio.com. The Core has two instruments housing 24 and 96 well capacity in order to optimize cell number and number of experimental conditions.

Traditional Respirometry (oxygen consumption rate)

The core houses traditional respirometers to measure oxygen consumption in samples that may not be suitable for Seahorse or may require more extensive experimental protocols. The Core houses an Oroboros Oxygraph2K as well as a traditional YSI Clark-type oxygen electrode. These respirometers are optimal for non-adherent samples such as muscle fibers and tissue homogenate.

Mitochondrial Reactive Oxygen Species Measurement

The Core is equipped to measure superoxide production by mitochondria in intact cells by MitoSOX fluorescence. Whole cell and isolated mitochondrial hydrogen peroxide production can also be measured in the Core by Amplex Red oxidation.

Electron Transport Complex Activity and Expression

The Core specializes in spectrophotometric kinetic enzymatic assays for the measurement of the enzymatic activities of the mitochondrial electron transport chain. Enzymatic activity can be supplemented with expression of the corresponding enzymes using Western Blot (antibodies and protocols validated in the Core). Activity and expression of the following enzymes can be measured:

Complex I
Complex II
Complex II/III
Complex IV
Complex V
Citrate Synthase

Apoptotic Signaling

The Core can measure aspects of the mitochondrial apoptotic cascade including mitochondrial swelling and PTP opening, caspase activation and cytochrome c release.

mtDNA/Nuclear DNA Ratios

Up to five prequalified TAQMAN sets can be used to determine mtDNA content (normalized to nDNA). Assays available for mouse, human, and chicken.
Up to three prequalified TAQMAN assays for mtDNA deletions as a marker of mitochondrial stress and replication dysfunction for mouse and human.
Precision mtDNA content or deletion load by dPCR for mouse and human samples.

Staff

Mike Reynolds

mjr122@pitt.edu
412-624-0462

Contact

Sruti Shiva, PhD | sss43@pitt.edu