Gas sensing in microplates with optodes: Influence of oxygen exchange between sample, air, and plate material

Arain, Sarina; Weiss, Svenja; Heinzle, Elmar; John, Gernot T.; Krause, Christian; Klimant, Ingo

doi:10.1002/bit.20348

Cited by 38 publications

(20 citation statements)

References 15 publications

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“…A model commonly used in in vitro studies is a static cell culture maintained at a constant external (atmospheric) pO 2 . For such a system, O 2 consumption rates and diffusion processes are important factors which contribute to O 2 transport to the cells from gaseous macro-phase 24,25 . Careful control of cellular O 2 in such experiments is still rare, and partial or complete deoxygenation and hypoxia-specific metabolic rearrangements may occur 17 and contribute to the observed biological effects.…”

mentioning

confidence: 99%

Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique

Zhdanov

Ogurtsov

Taylor

et al. 2010

Integrative Biology

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I.; Taylor, Cormac T.; et al. Publication date 2010 Publication informationIntegrative Biology, 2 (9): Publisher The Royal Society of ChemistryItem record/more information http://hdl.handle.net/10197/5029Publisher's version (DOI) 10.1039/c0ib00021cDownloaded 2019-04-05T04:17:49ZThe UCD community has made this article openly available. Please share how this access benefits you. Your story matters! (@ucd_oa) Some rights reserved. For more information, please see the item record link above. Running title: Oxygen Gradients in Respiring SamplesAbstract Changes in cellular O 2 levels elicit adaptive responses which can lead to the activation of oxygen-dependent transcription factors such as the hypoxia-inducible factor (HIF). However, our understanding of the determinants of these processes is still incomplete. Using the new intracellular O 2 sensing technique, we monitored O 2 gradients in static cultures of adherent PC12 cells exposed to graded atmospheric O 2 at rest and upon metabolic stimulation. Under high atmospheric O 2 (10-21%) the respiration of resting cells dictated that local O 2 was moderately reduced, and at a certain threshold (6% in galactose medium) cell layer became practically anoxic. Furthermore, cell stimulation triggered a major redistribution of O 2 and a prominent 'hypoxic overshoot' effect mediated by diffusion. The deep, prolonged cell deoxygenation upon stimulation was matched by an increase in nuclear HIF-1α levels. In the presence of nitric oxide the hypoxic overshoot was truncated and HIF-1α stabilization inhibited. Thus, the main determinants which impact upon cellular O 2 levels and oxygen-sensitive signaling pathways are the atmospheric O 2 , sample geometry, cell density, respiration rate and its dynamics. Changes in any of these parameters can significantly alter the O 2 levels experienced by the cells and the subsequently activated signaling pathways. A model commonly used in in vitro studies is a static cell culture maintained at a constant external (atmospheric) pO 2 . For such a system, O 2 consumption rates and diffusion processes are important factors which contribute to O 2 transport to the cells from gaseous macro-phase 24,25 . Careful control of cellular O 2 in such experiments is still rare, and partial or complete deoxygenation and hypoxia-specific metabolic rearrangements may occur 17 and contribute to the observed biological effects. In this work, using quenchedphosphorescence O 2 sensing technique which allows real-time monitoring of both intra-and extracellular O 2 concentration 22,26,27 we investigated O 2 gradients in dense populations of neurosecretory pheochromocytoma PC12 cells cultured and differentiated under standard conditions in 96-well plates (dPC12). dPC12 cells possess active oxidative phosphorylation and glycolysis, produce robust responses to excitatory stimulation 22,26 , and they represent a convenient model for studies of brain function and pathologies 28 . For such a system with resting respiring dPC12 cells, steady state O 2 gradients at different...

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confidence: 99%

Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique

Zhdanov

Ogurtsov

Taylor

et al. 2010

Integrative Biology

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“…High-resolution fluorescent imaging facilitates measuring changes in oxygen within individual cells, whereas the microplate-based systems are only applicable to large populations of cells (10 3 cells/well or greater in a sealed compartment) with end-point parameter readout (14). Furthermore, the kinetics of local changes in intracellular oxygen are much faster than the formation of global oxygen gradients in bulk sample, which eliminates the need for exclusion of ambient oxygen and sealing test samples (1). Thus, subtle and transient metabolic responses, such as those observed following CMC treatment of A549 cells, may be clearly seen by measuring intracellular oxygen but are outside the sensitivity of extracellular sensing methodologies.…”

Section: Discussionmentioning

confidence: 99%

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

O’Riordan¹,

Fitzgerald²,

Ponomarev³

et al. 2007

American Journal of Physiology-Regulatory, Integrative and Comp

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Papkovsky DB. Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging. Am J Physiol Regul Integr Comp Physiol 292: R1613-R1620, 2007. First published December 14, 2006; doi:10.1152/ajpregu.00707.2006.-The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptormediated Ca 2ϩ influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer. metalloporphyrin; mitochondrial function; uncoupling MOLECULAR OXYGEN IS A KEY substrate in aerobic biological systems, providing the terminal electron acceptor of the electron transport chain (7). The rate of oxygen consumption is closely regulated by the ATP/ADP ratio (24), while also being highly dependent on the concentration of available oxygen (31) and the action of signaling molecules such as NO (9, 37) and Ca 2ϩ (10, 23), which, in turn, are related to pathways of cell survival and death (22). Furthermore, the induction of cellular hypoxia results in the activation of a specific adaptive transcriptional response governed by the hypoxia inducible transcription factors (9). Oxygen consumption is, therefore, an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. It may be used to elucidate biochemical pathways of the cell and alterations in metabolism caused by various stimuli or disease states. Accordingly, there is a requirement for methods that can effectively monitor cellular oxygen levels and oxygen consumption rates.Optical schemes, based on the quenching by molecular oxygen of long-decay fluorescent and phosphorescent dyes, such as metalloporphyrins and ruthenium(II) complexes, have been under active development in recent years (32). The group of Wilson and coworkers (8, 47) ha...

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“…However, it is highly likely that, within the sample, micro-gradients of dissolved oxygen distribution may exist, from the top towards the bottom of the well or from the wall towards the centre of the well. This has recently been demonstrated in oxygen imaging experiments using fibreoptic oxygen microsensors which provide high spatial resolution 14 . Figure 3 shows processed data, where calculated slopes of the probe signal are related to cell concentration in test sample.…”

Section: Fluorescence Detection Mode and Assay Optimizationmentioning

confidence: 96%

Optical oxygen sensing systems for drug discovery applications: Respirometric Screening Technology (RST)

Papkovsky

Hynes²,

Fernandes³

2005

Chemical and Biological Sensors for Industrial and Environmental Security

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Quenched-fluorescence oxygen sensing allows non-chemical, reversible, real-time monitoring of molecular oxygen and rates of oxygen consumption in biological samples. Using this approach we have developed Respirometric Screening Technology (RST); a platform which facilitates the convenient analysis of cellular oxygen uptake. This in turn allows the investigation of compounds and processes which affect respiratory activity. The RST platform employs soluble phosphorescent oxygen-sensitive probes, which may be assessed in standard microtitter plates on a fluorescence plate reader. New formats of RST assays and time-resolved fluorescence detection instrumentation developed by Luxcel provide improvements in assay sensitivity, miniaturization and overall performance. RST has a diverse range of applications in drug discovery area including high throughput analysis of mitochondrial function; studies of mechanisms of toxicity and apoptosis; cell and animal based screening of compound libraries and environmental samples; and, sterility testing. RST has been successfully validated with a range of practical targets and adopted by several leading pharmaceutical companies.

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Gas sensing in microplates with optodes: Influence of oxygen exchange between sample, air, and plate material

Cited by 38 publications

References 15 publications

Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique

Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

Optical oxygen sensing systems for drug discovery applications: Respirometric Screening Technology (RST)

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