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Difference between revisions of "O2k-Specifications"

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|description='''O2k versus multiwell respirometer''': '''O2k''' stands for Oxygraph-2k and '''high-resolution respirometry''', meeting powerful quality criteria securing '''high output''' and pioneering state-of-the-art [[Gnaiger_2012_MitoPathways|OXPHOS analysis]] of substrate control and coupling control of mitochondrial function. 'High throughput' stands for '''disposable multiwell''' systems - expensive, with limited scope and extremely high running costs. In respirometry, high throughput is ''not equivalent to high output''.
|description='''O2k versus multiwell respirometer''': '''O2k''' stands for Oxygraph-2k and '''high-resolution respirometry''', meeting powerful quality criteria securing '''high output''' and pioneering state-of-the-art [[Gnaiger_2012_MitoPathways|OXPHOS analysis]] of substrate control and coupling control of mitochondrial function. 'High throughput' stands for '''disposable multiwell''' systems - expensive, with limited scope and extremely high running costs. In respirometry, high throughput is ''not equivalent to high output''.
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Revision as of 07:15, 16 February 2013

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O2k-Specifications

O2k-Catalogue

Description O2k versus multiwell respirometer: O2k stands for Oxygraph-2k and high-resolution respirometry, meeting powerful quality criteria securing high output and pioneering state-of-the-art OXPHOS analysis of substrate control and coupling control of mitochondrial function. 'High throughput' stands for disposable multiwell systems - expensive, with limited scope and extremely high running costs. In respirometry, high throughput is not equivalent to high output.
Product ID
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Type O2k, Feedback
Link
Image
caption


O2k versus multiwell respirometer

Comparison of the O2k and XFe

Authors: Gnaiger E, Fasching M, OROBOROS INSTRUMENTS, Innsbruck, Austria. Last edit: 2013-02-03


No single design is best for all. A specific respirometric instrument, therefore, cannot cover all applications in the best way. In this regard, the O2k and XFe are complementary. The two-chamber Oxygraph-2k can be extended to a Power-O2k system for combining high-resolution with high throughput. The 24- or 96-well XFe system is designed for high throughput screening. Qualitative and quantitative aspects are discussed below for comparison of the O2k and XFe. Scientists who have experience with both instruments are invited to extend the discussion.

>> Discussion: XFe for isolated mitochondria - a critical discussion - continued


Is plastic compatible with respirometry?

A. Oxygraph-2k
The O2k-Chambers are made of Duran glass and are closed by PVDF or PEEK stoppers which are as diffusion tight as titanium stoppers. The magnetic stirrer bars are coated by PVDF or PEEK, avoiding Teflon with high oxygen solubility (Gnaiger 1995). Viton O-rings are used for sealing the stoppers. Butyl rubber gaskets provide the seals for the oxygen sensors. These sealing materials minimize oxygen diffusion into or out of the experimental chambers.
The O2k not only minimizes the effect of oxygen backdiffusion by avoiding inappropriate plastic materials, but additionally implements automatic correction for instrumental background flux. Standardized protocols are available to evaluate and improve the accuracy of the instrumental background corrections. These instrumental tests are even performed automatically using standard programmes for feedback-control of the electronic Titration-Injection microPump, TIP2k.
>> Plastic materials with high oxygen diffusion are eliminated from the O2k-Chamber.
B. Multiwell
Oxygen storage in the plastic materials of multiwell plates leads to high oxygen backdiffusion. Since the problems are well known (Gnaiger 1995), specifications should be provided on oxygen backdiffusion, and test protocols should be applied to enable evaluation of such specifications (Gnaiger 2008).
At the high surface-to-volume ratio in a small well, the problem of plastic meterials is not restricted to oxygen diffusion. Lipid soluble substances (uncouplers, inhibitors) partition between the aqueous and plastic phases, such that the surface-attached biological sample is exposed to undefined effective concentrations.


Cost

How high are the running costs?

A. Oxygraph-2k
The running costs for the O2k are very low, as shown on the OROBOROS website and experienced by >400 users.
>> Low running costs for the OROBOROS O2k
B. Multiwell
The running costs are extremely high, based on expensive dischargeable wells for single use only.
How many of the wells of a dischargeable plate can actually be used for independent measurements? Several wells are required for calibration, edge effects may eliminate the use of wells on the sides. If more than four consecutive titrations are made, more wells are required for a single assay.
Elaborating a protocol for starting an experimental series requires a large number of test runs, such that the cost of discharged wells in an entire experiment approaches the investment into a second O2k.


Power-O2k - a 'best' investment

The OROBOROS O2k is not suited for high-throughput, with two experimental chambers. However, at the cost of an XFe (24 or 96 wells) several O2k instruments can be obtained. A large number of O2k chambers provides a unique high-throughput HRR system for quantitative O2k-measurements at low running costs.
>> Power-O2k.


Flexibility

MultiSensor versus multiwell

A. Oxygraph-2k: The modular concept of the O2k as a MultiSensor system
The O2k is designed as a flexible modular system. The O2k-Core supports add-on modules, for simultaneous measurement of oxygen flux and
* additional fluorometric measurement of ROS production, membrane potential, Ca2+ and ATP-production.
>> O2k-Fluorometry.
* additional potentiometric measurement of mitochondrial membrane potential (ion sensitive electrode: TPP+), Ca2+ in solution (using the same ion sensitive electrode after mounting a different membrane), and pH. An oxygen flux of 50 pmol/(s ml) corresponds - at an assumed O2 flux to extracellular H+ flux ratio of 1:1 - to a pH change of about 86 µpH/s in a very weak buffer (2 mM).
>> TPP+ and Ca2+; pH
* additional amperometric measurement of NO or H2S.
>> NO.
The DatLab software provides full flexibility for O2k-MultiSensor data monitoring.
B. Multiwell
The XFe is restricted to the additional measurement of pH. No specifications are given on sensitivity [µpH/s] of the measurement of acidification rate. Until recently, the effects of buffers including the bicarbonate system have been ignored. What is the drift of the pH signal?

Tissue preparations and cells

A. Oxygraph-2k
All tissue preparations including permeabilized muscle fibres, homogenate and isolated mitochondria can be studied in the O2k. Suspended blood cells and suspension cultures including yeast are ideally suited for the O2k. Monolayer cell cultures are trypsinized and studied in suspension. Neuronal cells may be studied attached to a disk inserted into the O2k (Brewer 2008 J Neurosci Methods, Jones 2009 Exp Neurol). Intact C. elegans is a perfect model for the O2k, whereas more delicate living animals, such as zooplankton, are likely to be put under improper stress in the stirred O2k-chamber.
>> O2k-Publications:_Specific_topics#Tissues_and_cell_types
B. Multiwell
Cells cultured in monolayer in the wells are the superior model for the XFe. Advertisements claim that all tissue preparations can be studied in the XFe. Permeabilized muscle fibres are seriously oxygen limited at oxygen levels at and below air saturation without stirring. Permeabilized cells may not remain attached to the wall and therefore impose a problem for the XFe technology, similar to tissue homogenate and isolated mitochondria.

OXPHOS analysis with multiple substrate-uncouplier-inhibitor titrations

A. Oxygraph-2k
Substrate-uncoupler-inhibitor titration (SUIT) protocols have been developed for OXPHOS analysis and high-resolution respirometry. This provides the basis for diagnostic tests of mitochondrial respiratory function, to study the complex interactions of coupling control and substrate control in a single assay, thus increasing the information obtained per unit sample and per unit time. More than 20 titration steps may be included in a single SUIT protocol.
>> O2k-Protocol: Substrate-uncoupler-inhibitor titration
B. Multiwell
The number of titrations into a well is limited to a maximum of four. The XFe, therefore, is not suited for application of SUIT protocols for OXPHOS analysis. Four titrations per well are insufficient for obtaining the information from a SUIT experiment. In this respect, the multiwell approach yields rather low throughput, since many wells are required for multiple titrations, and high inter-well variability represents an additional confounding factor.

Oxygen and temperature regime

A. Oxygraph-2k
The oxygen regime can be controlled for respiratory measurements for studies of hypoxia and hyperoxia in routine applications of the O2k. Oxygen kinetics of mitochondrial respiration is made possible by resolution of oxygen condentration in the nanomolar range.
At room temperature, experimental temperature can be constantly controlled in the range of 2 °C to 47 °C, at a temperature stability of +-0.001 °C. As a control, temperature and Peltier power are continuously measured and can be displayed any time.
B. Multiwell
Control of the oxygen regime is restricted in routine applications to intermittent equilibration of the unstirred medium with atmospheric oxygen and declining oxygen levels during measurement. Measurements at low oxygen levels are not possible due to very high oxygen backdiffusion, resulting in problems with zero oxygen calibration. The limit of detection is not specified. Incubation in gas controlled bench chambers is required for hypoxic or hyperoxic measurements.
Temperature stability and homogeneity between wells is a critical issue without being monitored. Experimental temperature cannot be regulated below room temperature.


Errors of measurement in respirometry and instrumental specifications

Quantitative respirometry

A. Oxygraph-2k
HRR is the result of long-term expertise in instrumental design, software development, and experimental protocols developed for mitochondrial physiology, clinical and pharmacological applications. Taken together, these developments resulted in new qualitative and quantitative standards summarized as the O2k-Concept.
The [1] specifications of the OROBOROS O2k are based on the many unique instrumental features:
  • Critical selection of materials yielding a nearly diffusion-tight chamber,
  • Long-term stability and linearity of the polarographic oxygen sensor (POS),
  • Highly automatic but transparent calibration routines and instrumental background correction,
  • Electronically controlled thermal environment with high temperature stability (+-0.001 °C),
  • Limit of detection of respiratory flux: +-1 pmol.s-1.ml-1.
B. Multiwell
From their basic design, multiwell systems are a tool for qualitative high-throughput screening, particularly for pharmacological testing. In many cases, results are not strictly quantitative, but merely relative changes are obtained. Huge oxygen fluxes reported by one group at a mitochondrial physiology meeting (MiPsummer School 2009, Baton Rouge, USA) were explained by a XFe representative as a "software problem". The absolute oxygen fluxes may represent artefacts. More recently, the problems of high oxygen diffusion were recognized, but the large corrections render non-specified errors in the calculation of background-corrected oxygen flux.


Accuracy of chamber volume and mixing

A. Oxygraph-2k
The O2k-chamber has a standard volume of 2 ml and is calibrated at an accuracy of better than +-1% (depending on calibrated pipettes), when inserting the stopper and filling the capillary at an error of <20 µl.
The entire effective volume (excluding the injection capillaries) is rigorously stirred.
B. Multiwell
No information is provided on the accuracy of the chamber volume in a multiwell system (~7 µl for the XF24). This inaccuracy translates directly to errors in the calculation of oxygen flux in the closed chamber. Similarly, accurate final concentrations of titrated substances are not known.
Mixing by moving the sensor/injector part up and down a few times is inadequate. Undefined diffusion layers develop during a measuring cycle.


How are cell number or mitochondrial protein defined?

A. Oxygraph-2k
In experiments with isolated mitochondria, tissue homogenates or suspended intact or permeabilized cells, the final concentration in the O2k-chamber is either defined by the preparation of the added suspension, and/or determined by taking a subsample from the chamber. In this way, the measured oxygen flux (per volume) can be expressed accurately per unit of biological sample (per mg protein, per million cells, etc.).
In experiments with permeabilized muscle fibers or other tissues, the tissue mass is determined before adding the sample into the O2k-chamber (e.g. 0.7 mg wet weight of mouse heart, 2 mg wet weight of human skeletal muscle), and the oxygen flux can then be expressed per tissue mass (mass-specific flux, reflecting mitochondrial density and functional quality).
The flexibility of the DatLab-software allows on-line display of respiratory flux per unit sample (per mg, or per Million cells) or per volume of the aqueous medium.
B. Multiwell
How many cells are actually enclosed in the compartment for measurement of respiration in a well, or which fraction of isolated mitochondria is outside versus inside the effective chamber? How can the recorded change in oxygen concentration be converted to respiration per million cells or per mg protein? Without solving these problems, no quantiative measurements of respiration are possible.


Are specifications comparable?

A. Oxygraph-2k
The limit of detection of oxygen flux is +-1 pmol O2 / (s ml). The limit of detection of oxygen concentration is 5 nmol/l (0.005 µM).
>> Detailed and unique specifications for the O2k.
B. Multiwell
No specifications are given on sensitivity (lower limit of detection of oxygen; non-linearity and restricted linear range; detection limit of oxygen flux) in some multiwell systems.


Summary

Quality versus quantity

A. Oxygraph-2k
The OROBOROS Oxygraph-2k for high-resolution respirometry (HRR) sets the gold standard for highly accurate quantitative measurements (which is high quality), following a scientific strategy. A new scientific level of OXPHOS analysis has been successfully introduced by SUIT protocols now widely applied with the O2k. High quality of instruments and methods is required in research and clinical applications. O2k-MultiSensor modules, particularly O2k-Fluorescence, extend HRR way beyond respirometry, making the O2k the most accurate and versatile instrument for cell respiration and OXPHOS analysis.
B. Multiwell
Lack of quality control of instrumental corrections yields artefacts that are published even in methodological articles on the XFe (high oxygen consumption at negative oxygen concentration). The maximum of four titrations per well possible with the XFe (i) prohibits necessary quality control in protocols requiring evaluation of saturating substrate concentration or optimum uncoupler concentration, leading to erroneous estimates of respiratory capacities, and (ii) limits OXPHOS analysis to the simplest protocols with restricted information.
>> XFe and published artefacts - a critical discussion.

Bioenergetics made simple

Scientific methods are developed and applied to help understanding cell metabolism. Opening new windows to a better understanding of cell metabolism requires a scientific enthusiasm and devotion to hard work beyond the easy ways of superficial plug-and-play approaches. Commmercial organizations advertise the XFe as making cell metabolism even easier. Scientific companies assist scientists instrumentally and methodologically, but do not make their subject (cell metabolism) more easy. Oxygen and pH - is this really cell metabolism revealed? Integration of catabolism and anabolism, ATP levels and ATP turnover, cell membrane and mitochondrial membrane potentials, redox states and intermediary metabolite levles, control of metabolic pathways - this and more is cell metabolism way beyond oxygen and pH.
Misleading simple interpretations: Coupling control in intact cells can be simply studied in a sequence of ROUTINE respiration (R), oligomycin-induced LEAK respiration (L), and evaluation of Electron transfer system capacity (E) (Hütter et al 2004). The R/E flux control ratio provides information on ROUTINE respiratory activity relative to ETS capacity (Gnaiger 2008, Gnaiger 2009). In commercially oriented (XFe) discussions, David Nicholls interprets the equivalent of the R/E flux control ratio as an index of reserve capacity. This is not supported by modern concepts on coupling control and is refuted by experimental evidence in human cell models and many tissue preparations (Gnaiger 2012 MitoPathways). This unjustified simplification (Bioenergetics made simple) ignores along the lines of outdated terminology (State 3 and State 3u) the fact that OXPHOS capacity (P) cannot in general be determined by noncoupled states of respiration (E). But E needs to be corrected for the P/E ratio if any apparent reserve capacity of ROUTINE respiration should be determined. This may still be considered as a very simple concept, but apparently not suffiently simple for XFe advertising.

Financial issues

The primary investment costs of the XFe system are tremendously high when compared with the Oxygraph-2k, particularly when comparing the limited scope of the XFe technology (limitation of titrations, limitations of MultiSensor extensions, limitation on quantification of results) with the O2k-Core, O2k-Fluorescence module and other OROBOROS MultiSensor modules.
The running costs of the O2k are uncomparably more economic than the high running costs of the XFe. The XFe running costs calculated over a single year cover the investement into a new O2k-Core plus its running costs.
Customers who can afford (mainly with governmental budgets) the running costs of the XFe will not need any discount on other less expensive and more efficient products.

Conflict of interest

Erich Gnaiger is founder and CEO of OROBOROS INSTRUMENTS, and responsible for the development of the Oxygraph-2k in collaboration with the O2k-Team and our partners. A series of methodological publications is the result of positive feedback between his work as faculty staff of the Medical University of Innsbruck, his company initiatives and numerous international cooperations.


MitoPedia methods: Respirometry