Talk:Setting the oxygen concentration

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

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

Increasing the oxygen concentration

Increase oxygen levels by adding H₂O₂ to a catalase containing medium MiR06

It is always possible to increase the oxygen concentrations by injecting H₂O₂ in the mitochondrial respiration medium MiR06 or MiR06Cr (MiR05 plus catalase) as described in MiPNet14.3. By using the TIP2k, the oxygen concentrations can be maintained between well defined limits, either using H₂O₂ or, for very low oxygen concentrations, air saturated medium in the TIP2k microsyringes. Such specification is called "oxystat" approach and supply appropriate templates for controlling the TIP2k. See also MiPNet12.10.

Add catalase to a final concentration of 280 IU/mL in the mitochondrial respiratory medium at the beginning of an experiment. 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 is not opened and closed and decreasing disturbance of the system and allowing faster 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. 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.

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 air saturation, as it 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 oxygen is injected into the gas phase of the O2k-Chamber.
Close the chamber when the O₂ concentration approaches the desired O₂ level.

Increase oxygen levels by opening the chamber

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 till oxygen concentration reaches an approximate level of the air calibration. Close the O2k-Chamber by closing the stoppers completely and wait till the POS is stable again (~ 5-10 minutes).

If possible, it is preferable to re-oxygenate in a phase of low measured respiratory activity 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) in the gas phase, followed by closing the stoppers once the desire oxygen level was reached, as described below:

Open chamber: Before injecting nitrogen, the O2k-Chamber needs to be opened to obtain a defined gas volume above the aqueous phase.
Using the 60 mL syringe N₂ is injected 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, when the "oxystat" follows.

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.