The Experts below are selected from a list of 3297 Experts worldwide ranked by ideXlab platform
Joel N Meyer - One of the best experts on this subject based on the ideXlab platform.
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seahorse xfe24 Extracellular Flux analyzer based analysis of cellular respiration in caenorhabditis elegans
Current protocols in immunology, 2015Co-Authors: Latasha L Smith, John P Rooney, Joel N MeyerAbstract:Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans. © 2015 by John Wiley & Sons, Inc. Keywords: mitochondrial toxicity; mitochondrial respiration; Seahorse XFe24; Caenorhabditis elegans
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Current Protocols in Toxicology - Seahorse Xfe24 Extracellular Flux Analyzer‐Based Analysis of Cellular Respiration in Caenorhabditis elegans
Current protocols in immunology, 2015Co-Authors: Latasha L Smith, John P Rooney, Joel N MeyerAbstract:Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans. © 2015 by John Wiley & Sons, Inc. Keywords: mitochondrial toxicity; mitochondrial respiration; Seahorse XFe24; Caenorhabditis elegans
Martin D Brand - One of the best experts on this subject based on the ideXlab platform.
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quantifying intracellular rates of glycolytic and oxidative atp production and consumption using Extracellular Flux measurements
Journal of Biological Chemistry, 2017Co-Authors: Shona A Mookerjee, David G Nicholls, Martin D Brand, Akos A GerencserAbstract:Abstract Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of Extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing differ- ent substrates. Mitochondrial P/O ratios are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel in- dices to quantify bioenergetic phenotypes. The Glycolytic Index reports the proportion of ATP production from glycolysis, and identifies cells as primarily glycolytic (Glycolytic Index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in Glycolytic Index, quantified by the Warburg Index. Additional indices quantify the acute flexibility of ATP supply: the Crabtree Index and Pasteur Index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the Supply Flexibility Index quantifies overall flexibility of ATP supply, and the bioenergetic capacity quantifies the maxi- mum rate of total ATP production. We illustrate the determination of these indices using C2C12 my- oblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how Extracellular Fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw Extracellular acidification and respiration data.
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determining maximum glycolytic capacity using Extracellular Flux measurements
PLOS ONE, 2016Co-Authors: Shona A Mookerjee, David G Nicholls, Martin D BrandAbstract:Measurements of glycolytic rate and maximum glycolytic capacity using Extracellular Flux analysis can give crucial information about cell status and phenotype during normal operation, development of pathology, differentiation, and malignant transformation. They are also of great use when assessing the effects of chemical or drug treatments. Here, we experimentally define maximum glycolytic capacity, demonstrate how it differs from glycolytic rate, and provide a protocol for determining the basal glycolytic rate and maximum glycolytic capacity in cells using Extracellular Flux measurements. The results illustrate the power of Extracellular Flux analysis to describe the energetics of adherent cells in culture in a fully quantitative way.
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Quantitative microplate-based respirometry with correction for oxygen diffusion.
Analytical Chemistry, 2009Co-Authors: Akos A Gerencser, David G Nicholls, David A Ferrick, Andy Neilson, Sung W. Choi, Ursula Edman, Nagendra Yadava, Richard J. Oh, Martin D BrandAbstract:Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O2 is consumed by the specimen, atmospheric O2 leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O2 but also store substantial amounts of gas. O2 Flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O2] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O2]. We optimize the algorithm to work with the Seahorse XF24 Extracellular Flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attac...
David A Ferrick - One of the best experts on this subject based on the ideXlab platform.
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the acute Extracellular Flux xf assay to assess compound effects on mitochondrial function
Journal of Biomolecular Screening, 2015Co-Authors: Ruolan Wang, David A Ferrick, Steven J Novick, James B Mangum, Kennedy L Queen, George W Rogers, Julie B StimmelAbstract:Numerous investigations have linked mitochondrial dysfunction to adverse health outcomes and drug-induced toxicity. The pharmaceutical industry is challenged with identifying mitochondrial liabilities earlier in drug development and thereby reducing late-stage attrition. Consequently, there is a demand for reliable, higher-throughput screening methods for assessing the impact of drug candidates on mitochondrial function. The Extracellular Flux (XF) assay described here is a plate-based method in which galactose-conditioned HepG2 cells were acutely exposed to test compounds, then real-time changes in the oxygen consumption rate and Extracellular acidification rate were simultaneously measured using a Seahorse Bioscience XF-96 analyzer. The acute XF assay was validated using marketed drugs known to modulate mitochondrial function, and data analysis was automated using a spline curve fitting model developed at GlaxoSmithKline. We demonstrate that the acute XF assay is a robust, sensitive screening platform f...
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Profiling metabolic and bioenergetic properties of 3D tumor microtissues (LB171)
The FASEB Journal, 2014Co-Authors: George W Rogers, Andy Neilson, Anne N Murphy, Brian P. Dranka, Pamela Swain, Ajit S. Divakaruni, David A FerrickAbstract:Cellular metabolism and bioenergetics processes are increasingly recognized as critical regulators in the pathogenesis of cancers. Metabolic activity and bioenergetic processes are often characterized using typical 2 dimensional (2D) adherent cell culture systems; however, this approach lacks the 3 dimensional (3D) context and heterogeneity of tumor tissue in vivo, which may have significant effects on metabolic and bioenergetic behavior. A novel and more physiologically relevant model is the use of microtissues (also known as spheroids). Metabolic activity and bioenergetic function were accessed in individual cancer cell line microtissues using a newly designed 96-well plate for the Seahorse Bioscience XFe96 Extracellular Flux Analyzer. Mitochondrial and glycolytic functions were determined simultaneously using the Cell Mito Stress and Glycolytic Stress Tests, respectively. Several parameters, including: seeding density, microtissue size, and substrate identity and availability were investigated. Further...
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Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform
Journal of Bioenergetics and Biomembranes, 2012Co-Authors: Sashi Nadanaciva, David A Ferrick, Craig C Beeson, Payal Rana, Gyda C. Beeson, Denise Chen, Yvonne WillAbstract:High-throughput applicable screens for identifying drug-induced mitochondrial impairment are necessary in the pharmaceutical industry. Hence, we evaluated the XF96 Extracellular Flux Analyzer, a 96-well platform that measures changes in the oxygen consumption rate (OCR) and Extracellular acidification rate (ECAR) of cells. The sensitivity of the platform was bench-marked with known modulators of oxidative phosphorylation and glycolysis. Sixteen therapeutic agents were screened in HepG2 cells for mitochondrial effects. Four of these compounds, thiazolidinediones, were also tested in primary feline cardiomyocytes for cell-type specific effects. We show that the XF96 platform is a robust, sensitive system for analyzing drug-induced mitochondrial impairment in whole cells. We identified changes in cellular respiration and acidification upon addition of therapeutic agents reported to have a mitochondrial effect. Furthermore, we show that respiration and acidification changes upon addition of the thiazoldinediones were cell-type specific, with the rank order of mitochondrial impairment in whole cells being in accord with the known adverse effects of these drugs.
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Quantitative microplate-based respirometry with correction for oxygen diffusion.
Analytical Chemistry, 2009Co-Authors: Akos A Gerencser, David G Nicholls, David A Ferrick, Andy Neilson, Sung W. Choi, Ursula Edman, Nagendra Yadava, Richard J. Oh, Martin D BrandAbstract:Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O2 is consumed by the specimen, atmospheric O2 leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O2 but also store substantial amounts of gas. O2 Flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O2] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O2]. We optimize the algorithm to work with the Seahorse XF24 Extracellular Flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attac...
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advances in measuring cellular bioenergetics using Extracellular Flux
Drug Discovery Today, 2008Co-Authors: David A Ferrick, Andy Neilson, Craig C BeesonAbstract:Cell-based assays have become a favored format for drug discovery because living cells have relevant biological complexity and can be highly multiplexed to screen for drugs and their mechanisms. In response to a changing Extracellular environment, disease and/or drug exposure, cells remodel bioenergetic pathways in a matter of minutes to drive phenotypic changes associated with these perturbations. By measuring the Extracellular Flux (XF), that is the changes in oxygen and proton concentrations in the media surrounding cells, one can simultaneously determine their relative state of aerobic and glycolytic metabolism, respectively. In addition, XF is time-resolved and non-invasive, making it an attractive format for studying drug effects in vitro.
Latasha L Smith - One of the best experts on this subject based on the ideXlab platform.
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seahorse xfe24 Extracellular Flux analyzer based analysis of cellular respiration in caenorhabditis elegans
Current protocols in immunology, 2015Co-Authors: Latasha L Smith, John P Rooney, Joel N MeyerAbstract:Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans. © 2015 by John Wiley & Sons, Inc. Keywords: mitochondrial toxicity; mitochondrial respiration; Seahorse XFe24; Caenorhabditis elegans
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Current Protocols in Toxicology - Seahorse Xfe24 Extracellular Flux Analyzer‐Based Analysis of Cellular Respiration in Caenorhabditis elegans
Current protocols in immunology, 2015Co-Authors: Latasha L Smith, John P Rooney, Joel N MeyerAbstract:Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans. © 2015 by John Wiley & Sons, Inc. Keywords: mitochondrial toxicity; mitochondrial respiration; Seahorse XFe24; Caenorhabditis elegans
Eugenia D Trushina - One of the best experts on this subject based on the ideXlab platform.
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comprehensive method for culturing embryonic dorsal root ganglion neurons for seahorse Extracellular Flux xf24 analysis
Frontiers in Neurology, 2012Co-Authors: Miranda Lange, Yan Zeng, Andrew M Knight, Anthony J Windebank, Eugenia D TrushinaAbstract:Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform.
