Oroboros O2K-FluoRespirometers

Oroboros O2K- is a state-of-the art respirometer for quantitative high-resolution respirometry and comprehensive OXPHOS analysis and combination of respirometry with fluorometry for simultaneous real-time monitoring of ROS production, mt- membrane potential, ATP production and Ca2+.

Mitochondrial energy metabolism is studied with the O2k in many research areas including exercise physiology, aging, cancer, diabetes, neurodegeneration, oxidative stress, and medicine. With the development of High-Resolution FluoRespirometry (HRFR), HRFR combines measurement and control of oxygen concentration and respiratory oxygen flux with monitoring ROS production, mitochondrial membrane potential, ATP production, Ca2+, pH, and NO – all in one chamber.

Features

The O2k is a flexible modular system that facilitates multiple possibilities to study multiple aspects of mitochondrial energy metabolism in mitochondria and cell research. The added support of the O2k-FluoRespirometer O2k-Module add-ons allows for simultaneous measurements of oxygen flux, fluorometric measurement of ROS production, mt- membrane potential (TMRM, safranin), Ca2+ or ATP-production, or potentiometric measurement of mt-membrane potential with TPP+ or TPMP+ (ion sensitive electrode, ISE), saponin (using the same ISE) or pH. All of this is supported by the Oroboros DatLab 7 software package that provides full flexibility for O2k-MultiSensor monitoring.

 

O2K measurements include:

  • Oxygen consumption
  • ROS production
  • Mitochondrial membrane potential
  • ATP production
  • Ca2+
  • pH
  • Nitric Oxide

O2K Sample types include:

  • Isolated mitochondria
  • Permeabilized tissues (e.g skeletal muscle fibres)
  • Intact cells
  • Permeabilised cells
  • Tissue homogenates

Specifications of HRR with the O2K FluoRespirometer

The specifications of the Oroboros O2k include several sole-source instrumental features integrated into a quality control system:

Specification  Detail
No. Chambers per O2K 2
Chamber volume 

2 mL (normal) and 0.5 mL (small) 

Oxygen Signal   
  • Noise at zero O2: <±0.02μM (SD, 100 data points recorded at 0.2s intervals at 37ºC) without smoothing (±0.005 μM typical)
  • Noise at air saturation: <±0.1μM O2 (SD, 100 data points recorded at 0.2s intervals at 37ºC) without smoothing at 180 μM O2 (±0.05 μM typical)
  • O2 range of linearity: 0 to 1000 µM.
  • Time constant: <4 s at 37 °C (<3 s typical).

Oxygen Flux, J02,v  [pmol.s-1.ml-1]

  • Limit of detection: 0.5 at steady-state over 5 min.
  • Sensitivity (normoxic): <1 at steady-state over 5 min at 20 40 °C.
  • Sensitivity (hyperoxic): <3 at steady-state over 5 min at 20-40 °C.
  • Noise: <0.2 after standard smoothing (120 s).
  • O2 range: flux measured up to 500 µM O2 (permeabilized fibres), and <0.1 µM based on DatLab analysis of oxygen kinetics (mitochondria and cells).

Instrumental background for linear measurements across the entire oxygen range

  • O2 backdiffusion [pmol.s-1.ml-1] at 0 µM: <3 at 20-40 °C (2.5 typical).
  • O2 consumption [pmol.s-1.ml-1] at 200 µM: <4 at 37 °C (3 typical); <3 at 25 °C (2 typical).

Temperature Stabilitiy 

  • Experimental temperature range: 4ºC to 47 ºC, recorded continuously
  • Temperature stability: ±0.002ºC  

DataLab Software 

  • Simultaneously display of O2 consumption and O2 flux
  • Barometric pressure for O2 calibration; temperature stability control
  • Automatic calibrations: O2 and O2K multi-sensor channels
DL-protocols: real-time guide through instrumental quality control tests and advanced diagnostic substrate-uncoupler-inhibitor titration (SUIT) protocols within a single assay for exploring mitochondrial function.     

Key Advantages 

  • Simple normalisation using cell number or mass/volume of tissue
  • Closed chambers
  • Validated for hypoxic assays 

Substrate-uncoupler-inhibitor titration (SUIT) protocols on the O2K

SUIT protocols have been developed for OXPHOS analysis and High-Resolution FluoRespirometry.  

SUIT protocols guide the user step-by-step through experiments which provide bioenergetic snapshots of mitochondrial fitness.

The flexibility of the O2k and SUIT protocols allows for any type of mitochondrial preparation to be measured: isolated mitochondria, permeabilized tissues, tissue homogenates, permeabilized cells, and intact cells.

SUIT protocols are the basis for O2K diagnostic testing for mitochondrial respiratory function and inform the study between complex interactions of coupling and substrate control in a single assay.

More than 20 titration steps can be included in a single SUIT protocol. 

O2K SUIT - Drosophila_2

Figure 1. DatLab data from a SUIT protocol measuring real-time respiration using homogenised drosophila.

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