O2k-Specifications

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O2k-Specifications

Description	O2k stands for Oxygraph-2k and high-resolution respirometry, meeting powerful quality criteria thus securing high output. 'High throughput' stands for multiwell systems. In respirometry, this is not equivalent to high output.
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Type	O2k, Feedback
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Comparison of HRR with other respirometric methods

• Review: Quantifying mitochondrial dysfunction in complex diseases of aging (Horan et al 2012)
• Comparison of common bioenergetic analysis methods (Zhang 2012 Nat Protoc)
• Discussion: Seahorse XF for isolated mitochondria - High throughput without high output? - Continue the discussion

Is plastic compatible with respirometry?

A. O2k

The O2k-durane glass chambers 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. Viton O-rings used for sealing of the stoppers, and butyl rubber gaskets provide the seals for the oxygen sensors.

The O2k not only minimizes the effect of oxygen backdiffusion by avoiding inappropriate plastic materials, but additionally implements automatic correction for instrumental background flux (see O2k-specifications).

>> Plastic materials with high oxygen diffusion are eliminated from the O2k-Chamber.

B. Multiwell

Oxygen storage in the plastic material of multiwell plates leads to high oxygen backdiffusion. Since the problems are well known (Gnaiger_1995_JBB), specifications should be provided on oxygen backdiffusion, and test protocols should applied to enable evaluation of such specifications (Gnaiger 2008).

At the high surface-to-volume ratio in a small well, the problem is not restricted to oxygen diffusion, but all lipid soluble substances partition between the aqueous and plastic phases, such that the surface-attached biological sample is exposed to undefined effective concentrations.

How high are the running costs?

A. O2k

The running costs for the OROBOROS O2k are very low.

>> 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. However, investing the identical amount of money, a large number of O2k chambers provides a unique high-throughput HRR system for quantitative O2k-measurements at low running costs.

>> Power-O2k.

Flexibility for MultiSensor applications

A. 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⁺, 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.

>> TPP⁺ and Ca²⁺; 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).

• additional amperometric measurement of NO or H₂S.

>> NO.

The DatLab software provides full flexibility for O2k-MultiSensor data monitoring.

B. Multiwell

The XF system 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?

Is OXPHOS analysis with multiple substrate-uncouplier-inhibitor titrations possible?

A. O2k

Substrate-uncoupler-inhibitor titration (SUIT) protocols have been developed for OXPHOS analysis. 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, such that modern SUIT protocols cannot be applied in multiwell systems. Four titrations per well are insufficient for obtaining the information from a SUIT experiment. In this respect, the multiwell approach yields rather low throughput.

Quantitative respirometry

A. O2k

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 company 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. O2k

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. O2k

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 in the closed 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. O2k

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

The OROBOROS Oxygraph-2k for high-resolution respirometry (HRR) is the gold standard for highly accurate quantitative measurements, using low amounts of sample. Instrumental and methodological approaches require high quality in research and clinical applications.

Lack of quality control yields publications which may pass the scientific reviewing system without meeting basic scientific quality criteria. For a discussion of a publication based on a multiwell system (Rogers et al 2011 PLoS One), see >> Discussion: HRR versus multiwell.

MitoPedia methods: Respirometry