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Cellular Bioenergetics (XF24-3 Analyzer)

XF instrument

Overview

The Seahorse XF24-3 Extracellular Flux Analyzer allows for real-time quantitation of two major energy-yielding pathways in the cell: mitochondrial respiration and glycolysis. This instrument offers considerable improvements compared to former methodologies. XF24-3 analyzers do not require radiolabeled substrates, and unlike the classic Clark electrode, it is possible to make high-throughput measurements of cellular oxygen consumption on 24 samples simultaneously in a microplate format.

The XF24-3 uses disposable fluorescent-based optical sensors to measure oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and carbon dioxide production rate (COPR) directly in the media of culture microplates. OCR and ECAR reflect the flux through mitochondrial oxidative metabolism and glucose oxidation, respectively. Changes in cellular metabolism in response to specific inhibitors or other substances can also be determined. Built-in injection ports surrounding each of the culture wells in a microplate allow sequential delivery of up to four compounds (e.g., fatty acids, glucose, 2,4-dinitrophenol, FCCP, oligomycin) to cells during the course of an experiment.

An XF Islet Capture Microplate is available to assess whole islet bioenergetics in vitro (30-70 islets per well, for glucose or oligomycin response studies). It is possible to use this kit to assess respiration from tissue biopsies.

The XF Technology:

  • Measures oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and carbon dioxide production rate (COPR) of living cells simultaneously in real time.
  • Completely non-invasive measurement requires no addition of dyes, labels or reporters.
  • Cell types assayed on the XF analyzer: Cell lines, primary cells, isolated mitochondria, small tissue pieces (pancreatic islets, tissue biopsies from animals).
  • Typically requires only 30,000 to 80,000 cells per well in 24-well plates.
  • Cells and plates are not affected by XF measurement and may be used for an additional assay.
  • Measurements may be repeated multiple times to measure kinetic responses.
  • No cleaning required. All parts that contact cells, media or drugs are disposable.
  • Measures adherent cells without requiring trypsinization.
  • Up to four test compounds (drugs or substrates) may be added automatically to each well. Measurements are performed before and after each compound is added.
assistance

Contact Laurent Vergnes at lvergnes(at)ucla.edu for potential collaborative opportunities.

Laboratory of Karen Reue | Human Genetics | David Geffen School of Medicine | UCLA