Extracellular Flux Analysis to Monitor Glycolytic Rates and Mitochondrial Oxygen Consumption

Pelletier, Martin; Billingham, Leah K.; Ramaswamy, Madhu; Siegel, Richard M.

doi:10.1016/b978-0-12-416618-9.00007-8

Cited by 80 publications

(85 citation statements)

References 81 publications

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“…21 Briefly, NK cells are immobilised within the XF base medium supplemented with a final concentration of 10 mM glucose, 1 mM sodium pyruvate and 2 mM L-glutamine. Medium was warmed in a water bath at 37°C, before pH adjustment to 7.4±0.1 using 1N NaOH.…”

Section: Mitochondrial Stress Assaymentioning

confidence: 99%

“…21 Briefly, NK cells were immobilised in Glycolytic stress medium was comprised of XF base medium supplemented with a final concentration of 2 mM L-glutamine. pH was adjusted to 7.4± 0.1 using 1N NaOH in a 37°C water bath.…”

Section: Bhi = Log (Spare Repiratory Capacity)×(atp Production) (Non-mentioning

confidence: 99%

“…Mitochondrial respiration has been reported as oxygen consumption rate (OCR) at pmol/min. 21 Bioenergetic health index (BHI) was used to measure the NK cells overall mitochondrial biogenetic function and was calculated based on the formula below. Shapiro-Wilk analysis and Leven's test were used to determine normality and homogeneity.…”

Section: Supplement Figurementioning

confidence: 99%

See 2 more Smart Citations

Reduced glycolytic reserve in isolated natural killer cells from Myalgic encephalomyelitis/chronic fatigue syndrome patients: A preliminary investigation

Nguyen

Staines

Johnston

et al. 2018

Asian Pac J Allergy Immunol

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Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is medically unexplained post-exertional fatigue associated with significant reduction in natural killer cell (NK) cytotoxicity activity. Cytotoxic activity relies on glycolytic flux and mitochondrial respiration to fulfill energetic cellular demands. While mitochondrial dysfunction has been reported in ME/CFS patients, no previous investigation has examined the bioenergetic profile of isolated NK cells from ME/CFS patients.

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Section: Mitochondrial Stress Assaymentioning

confidence: 99%

Section: Bhi = Log (Spare Repiratory Capacity)×(atp Production) (Non-mentioning

confidence: 99%

See 1 more Smart Citation

Reduced glycolytic reserve in isolated natural killer cells from Myalgic encephalomyelitis/chronic fatigue syndrome patients: A preliminary investigation

Nguyen

Staines

Johnston

et al. 2018

Asian Pac J Allergy Immunol

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“…However, it is clear in multiple other systems that CO 2 can be a major contributor to extracellular acidification [4][5] . Multiple papers acknowledge this contribution, but do not attempt direct quantitation of CO 2 -derived acid 3,8,9 . We recently demonstrated quantitatively that CO 2 production is a significant source of extracellular acidification in this system 6 .…”

Section: Introductionmentioning

confidence: 99%

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Mookerjee

Brand

2015

JoVE

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Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis 1 . Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate 2 , the same is not true for extracellular acid production and its relationship to glycolytic rate [3][4][5][6] . Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO 2 , produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate -+ H + and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO 2 , hydration to H 2 CO 3 and dissociation to HCO 3 -+ H + is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6 . Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

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“…Real time, simultaneous measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), as indicators of oxidative phosphorylation and glycolysis, is a common strategy to address the states of energy producing pathways [11][12][13] . In order to achieve this, an extracellular flux analyzer, such as the Seahorse XF 96, is routinely used.…”

Section: Introductionmentioning

confidence: 99%

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Traba

Miozzo

Akkaya

et al. 2016

JoVE

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Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells. Video LinkThe video component of this article can be found at

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Extracellular Flux Analysis to Monitor Glycolytic Rates and Mitochondrial Oxygen Consumption

Cited by 80 publications

References 81 publications

Reduced glycolytic reserve in isolated natural killer cells from Myalgic encephalomyelitis/chronic fatigue syndrome patients: A preliminary investigation

Reduced glycolytic reserve in isolated natural killer cells from Myalgic encephalomyelitis/chronic fatigue syndrome patients: A preliminary investigation

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

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