Description: Overview of Core

The XF24-3 is a unique instrument that allows quantitation of cellular energetic pathways in cultured cells in real time. The XF24-3 quantifies the activity of the two major energy-yielding pathways in the cell, mitochondrial respiration and glycolysis. The XF24-3 instrument provides a significant advance over existing methodologies to quantitate cellular metabolic pathways. Unlike radiometric assays, the XF24-3 does not require radiolabeled substrates, and unlike measurements of cellular oxygen consumption using the classic Clark electrode, it is possible to make high throughput measurements on 24 samples simultaneously in a microplate format. The XF24-3 uses disposable fluorescent-based optical sensors to measure oxygen consumption rate (OCR), carbon dioxide production rate (COPR), and extracellular acidification rate (ECAR) directly in the culture media of culture microplates. These values inform about the flux through mitochondrial oxidative metabolism (OCR) and glucose oxidation (ECAR). In addition, through built-in injection ports surrounding each of the culture wells in the microplate, it is possible to sequentially deliver up to 4 compounds (for example, fatty acid, glucose, 2,4-dinitrophenol, FCCP, oligomycin) to the cells during the course of an experiment to determine changes in cellular metabolism in response to specific inhibitors or other compounds.

NEW: A XF Islet Capture Microplate is available to assess whole islet bioenergetics in vitro (30-70 islets per well, glucose or oligomycin response studies). There is a possibility 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.

Examples of experimental output:

Pricing through recharge, PO, check or credit card. User options include 3 levels of involvement by Core personnel. Appointment only.

Premium Service. $263.29 per essay

• Users will provide information concerning goal of experiment and cells in XF24 plates.
• Core facility will provide with proper experimental design and provide all XF consumables.
• Users will plate and grow cells or tissues for XF assay.
• Core facility will perform media exchange, prepare XF cartridge with compounds, create XF assay template and run assay on XF24-3 instrument.
• Core facility will perform data analysis and generate data summary.

Regular Service. $159.51 per essay

Basic Service. $120.59 per essay

• Users will perform all steps from experimental design to data analysis.
• Users will provide XF consumables.
• Require approval for independent operation of the XF24-3 instrument

Consultation and collaboration are available. Please contact Dr. Laurent Vergnes or Dr Karen Reue.

Resources:

The Seahorse Bioscience company website: http://www.seahorsebio.com

Reviews

• Integration of cellular bioenergetics with mitochondrial quality control and autophagy. Hill BG, Benavides GA, Lancaster JR Jr, Ballinger S, Dell'Italia L, Jianhua Z, Darley-Usmar VM. J Biol Chem. 2012 Dec;393(12):1485-1512.
• Advances in measuring cellular bioenergetics using extracellular flux. Ferrick DA, Neilson A, Beeson C. Drug Discov Today. 2008 Mar;13(5-6):268-74.

For mitochondrial essay (cell line):

• Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes. Bordicchia M, Liu D, Amri EZ, Ailhaud G, Dessì-Fulgheri P, Zhang C, Takahashi N, Sarzani R, Collins S. J Clin Invest. 2012 Mar 1;122(3):1022-36.
• UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells. Zhang J, Khvorostov I, Hong JS, Oktay Y, Vergnes L, Nuebel E, Wahjudi PN, Setoguchi K, Wang G, Do A, Jung HJ, McCaffery JM, Kurland IJ, Reue K, Lee WN, Koehler CM, Teitell MA. EMBO J. 2011 Nov 15;30(24):4860-73.
• Paraoxonase 2 deficiency alters mitochondrial function and exacerbates the development of atherosclerosis. Devarajan A, Bourquard N, Hama S, Navab M, Grijalva VR, Morvardi S, Clarke CF, Vergnes L, Reue K, Teiber JF, Reddy ST. Antioxid Redox Signal. 2011 Feb 1;14(3):341-51.

For glycolysis and fatty acid oxidation essays:

• Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA. Am J Physiol Cell Physiol. 2007 Jan;292(1):C125-36.
• Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance. Vergnes L, Chin R, Young SG, Reue K.. J Biol Chem. 2011 Jan 7;286(1):380-90.
• Zbtb16 has a role in brown adipocyte bioenergetics Plaisier C L, Bennett B J, He A, Guan B, Lusis A J, Reue K and Vergnes L. Nutrition and Diabetes 2. 2012 Sep 17;2:e46.
• Free base lysine increases survival and reduces metasttasis in protate cancer model. Ibrahim-Hashim A, Wojtkowiak JW, de Lourdes Coelho Ribeiro M, Estrella V, Bailey KM, Cornnell HH, Gatenby RA, Gillies RJ. 2011. Suppl 1(4). pii: JCST-S1-004.

For isolated mitochondria assays:

• High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. Rogers GW, Brand MD, Petrosyan S, Ashok D, Elorza AA, Ferrick DA, Murphy AN. PLoS One. 2011;6(7):e21746.
• Mutant Twinkle increases dopaminergic neurodegeneration, mtDNA deletions and modulates Parkin expression. Song L, Shan Y, Lloyd KC, Cortopassi GA..Hum Mol Genet. 2012 Dec 1;21(23):5147-58.
• Secreted frizzled-related protein 5 suppresses adipocyte mitochondrial metabolism through WNT inhibition. Mori H, Prestwich TC, Reid MA, Longo KA, Gerin I, Cawthorn WP, Susulic VS, Krishnan V, Greenfield A, Macdougald OA. J Clin Invest. 2012 Jul 2;122(7):2405-16.

For respiration in tissues

• Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance. Vergnes L, Chin R, Young SG, Reue K. J Biol Chem. 2011 Jan 7;286(1):380-90.
• Acute stimulation of white adipocyte respiration by PKA-induced lipolysis. Yehuda-Shnaidman E, Buehrer B, Pi J, Kumar N, Collins S. Diabetes. 2010 Oct;59(10):2474-83.
• ABCC6 Localizes to the Mitochondria-Associated Membrane. Martin LJ, Lau E, Singh H, Vergnes L, Tarling EJ, Mehrabian M, Mungrue I, Xiao S, Shih D, Castellani L, Ping P, Reue K, Stefani E, Drake TA, Bostrom K, Lusis AJ. Circ Res. 2012 Aug 17;111(5):516-20.