High-Resolution Respirometry for Permeabilized Tissues Using Oroboros
Rationale: Measures of mitochondrial respiratory function.
Instrument: Oxygraph-2K (Oroboros Instruments).
Measures: Measures of respiratory activity (oxidative phosphorylation in mitochondria) of biological samples and quantitative data are obtained.
Data Inference: Oxygen concentration and flux.
Controls: Care is taken to maintain the linearity, stability and sensitivity of the signal from polarographic oxygen sensors.
Mitochondrial Function in Primary Cell Cultures Using Seahorse
Rationale: Measures of mitochondrial function in primary cell cultures.
Instrument: XF24 Extracellular Flux Analyzer (Seahorse Instrument).
Measures: In vitro oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), indicative of mitochondrial activity and fatty acid oxidation are measured.
Data Inference: Data are dissolved oxygen and pH, which reflect OCR and ECAR.
Control: Care is taken that the biological samples tested and the running buffer is acclimatized to neutral pH at 37oC prior to running the assay.
ATP Production, Membrane Potential and ROS Release in Isolated Mitochondria
Rationale: Measures of ATP production, membrane potential and ROS release in isolated mitochondria.
Instrument: Perkin Elmer Victor X Multimode Plate Reader.
Measures: Absorbance, fluorescence and UV capability are measured using multiple-assay technology.
Data Inference: Data are measurements of mitochondrial ATP production rate, reactive oxygen species production rate, and mitochondrial membrane potential.
Control: Care is taken to control and monitor temperature as necessary for assay conditions.