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Setting the oxygen concentration

From Bioblast


O2k-Open Support

Setting the oxygen concentration

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O2k-Open Support

MitoPedia O2k and high-resolution respirometry: O2k-Open Support 

The oxygen concentration can be increased or decreased during an instrumental or a biological experiment. Increasing or decreasing oxygen concentration during an experiment with a biological sample is relevant for specific applications (e.g., hypoxia, tissue normoxia, high oxygen levels for working with permeabilized muscle fibers) or simply if an experiment runs out of oxygen before it is finished (re-oxygenations). There are different possibilities to adjust oxygen concentration in the Oroboros O2k:

Increasing the oxygen concentration

It is always possible to increase the oxygen concentration by injecting H2O2 into the mitochondrial respiration medium MiR06 (MiR05 plus 280 IU/mL catalase) or MiR06Cr (MiR06 plus 20 mM creatine) as described in MiPNet14.3. By using the TIP2k, the oxygen concentrations can be maintained within well defined limits using H2O2 in the TIP2k microsyringes. This method is called the "oxystat" approach and supplies an appropriate template for using the TIP2k to control oxygen concentrations. See also the Feedback Control Mode Section in MiPNet12.10.
When oxygen starts to become limited, inject 1-3 µL of 200 mM H2O2 stock solution. The H2O2 is quickly converted to O2 with a high catalase concentration able to avoid any sample oxidative stress. During this procedure the O2k-Chamber remains closed, decreasing disturbance of the system and allowing for quicker POS stabilization.
The initial increase in oxygen, however, is preferentially made by injecting oxygen, since there is the risk of bubble formation if the oxygen concentration is increased in a single large step (see below). If oxygen gas is not available for the initial oxygenation, a very small bubble may be left in the chamber while slowly rising the oxygen level to 500 µM with additions of H2O2, such that gas can escape into the small bubble and then be extruded by fully closing the chamber. During the experiment re-oxygenations must be progressively performed in order to avoid gas bubble formation.
*The mitochondrial respiration medium MiR06 contains catalase. If the respiration medium MiR05 is being used, 5 µL of catalase stock (final concentration of 280 IU/mL) can be added to to allow re-oxygenations with H2O2.
  • Increase oxygen levels with injection of oxygen into the gas phase
If you replace the gas phase above the liquid phase by pure oxygen you can increase oxygen levels above normal air saturation, as is recommended for measuring mitochondrial respiratory function in muscle biopsies.
  • Open chamber: Before injecting oxygen, the O2k-Chamber needs to be opened to obtain a defined gas phase above the aqueous phase.
  • Using the 60 mL syringe inject oxygen into the gas phase of the O2k-Chamber.
  • Close the chamber when the O2 concentration approaches the desired O2 level.
Lift the stopper using the Stopper-Spacer tool to a standard position with a fixed gas phase above the aqueous phase in the O2k-Chamber. Leave the O2k-Chamber open until the oxygen concentration reaches an approximate level of the air calibration. Close the O2k-Chamber by closing the stoppers completely and wait until the POS is stable again (~ 5-10 minutes).
If possible, it is preferable to re-oxygenate in a phase where respiratory activity measurements are low, since minor amounts of oxygen are consumed during the stabilization phase of the POS after closing the O2k-Chamber.

Decreasing the oxygen concentration

Low oxygen levels can be reached depending on the application. During an experiment with a biological sample this may be performed by injection of nitrogen (N2) or hydrogen (H2) into the gas phase, followed by closing the stoppers once the desire oxygen level is reached, as described below:
  • Open chamber: Before injecting N2 or H2, the O2k-Chamber needs to be opened to obtain a defined gas volume above the aqueous phase.
  • Using the 60 mL syringe inject N2 or H2 into the gas phase of the O2k-Chamber.
  • Close the chamber when the O2 concentration approaches the desired O2 level.
However, often the desired starting value is simply reached by waiting until the sample has consumed the required amount of oxygen.
To perform zero calibration oxygen should be removed by the addition of dithionite or biological sample.
To reduce the oxygen levels during an instrumental O2 background test, dithionite should be used.