Technologies

Seahorse Extracellular Flux Analyzer (XFe24)

Measures O2 consumption rate (OCR) and extracellular acidification rate (ECAR) of adherent cells, in a 24-well format. Recent modeling and computational advances that correct for O2 fluxes unrelated to cellular O2 reduction have resulted in a further increase in sensitivity such that many human primary cells that have low O2 consumption rates can now be reliably studied. Recent advances in experimental design have supplied protocols that allow the ECAR signal to be specific for glycolytic flux. The instrument has been widely endorsed by the bioenergetics community. No other method for adherent cells approaches the sensitivity of the Seahorse instrument.

TYPE of Samples

• Adherent cells
• Isolated mitochondria
• Zebrafish embryos
• Some tissue preps (need for substantial optimization should be expected)

Specifications

• Consumables: Custom cell culture plate and sensor cartridge, only available from Agilent
• Up to 4 injections during an experiment.
• Experimental temperature range: 16 – 42°C

Additional Details

Some information, including details about the technology and some protocols (Agilent): https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-analyzers

Consumables (Agilent):
https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-sensor-cartridges-cell-culture-microplates

Instrument availability:
One instrument is available for use as part of the MitoCare Shared Resources service center.

Oroboros Oxygraph 2K

Measures O2 consumption rate of samples in suspension. Typical samples are isolated mitochondria and cells. The instrument has a two-sample capacity. Its high degree of sensitivity makes it state-of-the-art. The instrument can also be adapted for parallel determinations of O2 consumption and mitochondrial inner membrane potential using a TPMP+ electrode.

OXYMax Indirect Calorimetry System

Measures whole-body O2 consumption and CO2 production. Equipment is housed in a light-cycle controlled dedicated room, with monitoring of room humidity and temperature.

Exercise & Behavior

Rodent treadmill “Exer 3/6” (Columbus Instr.)

Software-controlled for speed, acceleration, and incline. The software also records the distance traveled. Equipment is housed in a dedicated room.

Metabolic modular treadmill for mice (Columbus Instr.)

A one-lane treadmill with the same features as the ‘Rodent Treadmill’, but which can be enclosed with air-tight seals for measuring oxygen consumption during animal exercise through coupling with the OXYMAX indirect Calorimetry System.

Muscle force measurements

1. Standalone ASI contractile force measurement equipment for recording skeletal muscle force in vivo or in isolated skeletal muscle during electrical stimulation.
2. Microscope adaptable, ASI force transducer and electrical stimulation unit to record force during isometric contraction of skeletal muscle fibers/cardiac trabeculae simultaneously with microscopic observation.

Rotarod (Maze Engineers)

Widely used behavioral task to evaluate motor performance. The instrument can be customized for speed, acceleration, and is controlled using the Conductor Software.

Murine Echocardiography

Multiwell Reader

BIOTEK Synergy neo2

High-end plate reader that measures fluorescence, absorbance, and luminescence. The instrument is designed with separate optics for each of these capabilities; thus each capability has been optimized. The instrument also allows dual-wavelength excitation, and time-resolved fluorescence, and is equipped with an injection system. The instrument can also be programmed to read from a custom Seahorse microplate.

Proteomics

Mitochondrial Mass Spectrometry

Several investigators at MitoCare use mass spectrometry approaches to interrogate the protein composition of different mitochondrial compartments: from mitochondrial DNA nucleoids to the constituents of the mitochondrial-endoplasmic reticulum contact sites. Proteins are purified from different biological samples, in their native state or after isotope tagging (SILAC) or proximity biotinylation.

Gene Profiling

Mitochondrial DNA Expression

An indirect reading of the metabolic capacity of a cell or a tissue is provided by the total amount of mitochondrial DNA (mtDNA) present at any given time, its transcription rate, and the proportion of mutated versus wild-type molecules. We developed and optimized a wide range of assays to evaluate mtDNA copy number (in human and mouse samples) and mutation (i.e. deletion) using real-time quantitative PCR coupled to relative or absolute quantification strategies. Similarly, specific assays are available for mitochondrial gene transcription. 

• Chronic Alcoholic Rat
• High Fat Diet Mouse
• Friedreich's Ataxia
• PiC Knock-Out
• MICU1 Knock-Out<
• MICU2 Knock Out
• Miro1 Knock Out
• IP3r Knock-Out
• EMRE/Smtd1 Knock-Out
• ACOT2 Knock-Out
• DRP1 Knock-Out

• Fibroblasts and Lymphoblasts
• PiC Loss of Function
• MICU1 and MICU2 Loss of Function
• Autosomal Dominant Optic Atrophy OPA1 mutations
• MSTO1 mutations
• MERRF and MELAS Fibroblasts
• "Common Deletion" Fibroblasts