Difference between revisions of "Talk:Setting the oxygen concentration"

Revision as of 09:45, 9 June 2020

Talk:Setting the oxygen concentration

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

The oxygen concentration can be increased or decreased during an experiment with biological samples, depending on the application (e.g., for achieving hypoxia, with permeabilized muscle fibers, etc) or simply whenever oxygen concentrations run low before the end of an experiment. There are different means to adjust oxygen concentrations:

Increasing the oxygen concentration

It is always possible to increase the oxygen concentrations by combining catalase and injecting H₂O₂ in the mitochondrial respiratory medium 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.

Increase oxygen levels by combining H₂O₂ to a catalase containing medium (Medium-MiR06)

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 should not be opened and closed to decrease disturbance of the system and to allow a faster POS stabilization.

Increase oxygen levels with H₂O₂ injections small volume (µL) of 200 mM H₂O₂ stock solution is injected into the O2k-Chamber filled with MiR06.

With MiR06 (or MiR06Cr), the medium in the O2k-Chamber can be re-oxygenated very conveniently with H₂O₂ injections. 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 volume above the aqueous phase.
Using the 60 mL syringe pure 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. (1) during an experiment with a biological sample this may be performed by injection of N₂ in the gas phase, followed by closing the stoppers once, as described in Nitrogen injection. 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.

For calibration and for instrumental O₂ background tests purposes, dithionite is used to reduce oxygen levels, however this is not recommended in the presence of biological samples.

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 {{Technical support}}
-The oxygen concentration can be increased or decreased during an experiment with biological sample, depending on the desire approach (hypoxia, working with muscle fibers...) or simply if the experiment is not done and the oxygen went low.
+The oxygen concentration can be increased or decreased during an experiment with biological samples, depending on the application (''e.g.,'' for achieving hypoxia, with permeabilized muscle fibers, etc) or simply whenever oxygen concentrations run low before the end of an experiment. There are different means to adjust oxygen concentrations:
 === Increasing the oxygen concentration ===
-It is always possible to increase the oxygen concentration using the combination of catalase in the medium and injections of H2O2, as described in  [[MiPNet14.13 Medium-MiR06| MiPNet14.3]]. By using the [[TIP2k]] the oxygen concentration can be maintained between well defined limits, either using H2O2 or (for very low oxygen concentrations) air saturated medium in the TIP syringes. We call this an "oxystat" approach and supply appropriate templates for controlling the TIP2k. See also [[MiPNet12.10]].
+It is always possible to increase the oxygen concentrations by combining catalase and injecting H<sub>2</sub>O<sub>2</sub> in the mitochondrial respiratory medium as described in  [[MiPNet14.13 Medium-MiR06| MiPNet14.3]]. By using the [[TIP2k]], the oxygen concentrations can be maintained between well defined limits, either using H<sub>2</sub>O<sub>2</sub> or, for very low oxygen concentrations, air saturated medium in the [https://www.bioblast.at/index.php/Microsyringe%5C500_mm3%5CTIP2k TIP2k microsyringes]. Such specification is called "oxystat" approach and supply appropriate templates for controlling the TIP2k. See also [[MiPNet12.10]].
+*'''Increase oxygen levels by combining H<sub>2</sub>O<sub>2</sub> to a catalase containing medium (Medium-MiR06)'''
-An experiment needs not necessarily be terminated, because of running out of oxygen. There are different possibilities to re-oxygenate.
+: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 H<sub>2</sub>O<sub>2</sub> stock solution. The H<sub>2</sub>O<sub>2</sub> is quickly converted to O<sub>2</sub> with a high catalase concentration able to avoid any sample oxidative stress. During this procedure the O2k-Chamber should not be opened and closed to decrease disturbance of the system and to allow a faster POS stabilization.
-* '''To increase oxygen levels''' small volumes (µL) of [[Reoxygenation#Preparation_of_200_mM_H2O2_stock_solution|200 mM H<sub>2</sub>O<sub>2</sub> stock solution]] are injected into the O2k-chamber filled with 2 mL MiR06.
-With MiR06 (or [[MiR06Cr]]), the medium in the O2k-chamber can be re-oxygenated very conveniently with H<sub>2</sub>O<sub>2</sub> titrations. The initial increase in oxygen, however, is preferentially made with oxygen gas, 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 H<sub>2</sub>O<sub>2</sub>, such that gas can escape into the small bubble and then be extruded by fully closing the chamber. During the experiment, re-oxygenations are sufficiently small such that H<sub>2</sub>O<sub>2</sub> titrations into the closed chamber do not lead to gas bubble formation.
+* '''Increase oxygen levels with H<sub>2</sub>O<sub>2</sub> injections''' small volume (µL) of [[Reoxygenation#Preparation_of_200_mM_H2O2_stock_solution|200 mM H<sub>2</sub>O<sub>2</sub> stock solution]] is injected into the [[O2k-Chamber]] filled with MiR06.
-* '''Increase oxygen levels with pure oxygen'''
+With MiR06 (or [[MiR06Cr]]), the medium in the O2k-Chamber can be re-oxygenated very conveniently with H<sub>2</sub>O<sub>2</sub> injections. 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 H<sub>2</sub>O<sub>2</sub>, 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.
-If you replace the air 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 gas, the O2k-Chamber needs to be opened to obtain a defined gas volume above the aqueous phase.
+* '''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 volume above the aqueous phase.
 :* Using the[[Syringe\60 ml\Gas-Injection | 60 mL syringe]] pure oxygen is injected into the gas phase of the O2k-Chamber.
 :* Close the chamber when the O<sub>2</sub> concentration approaches the desired O<sub>2</sub> level.
-*'''Re-oxygenation by adding H<sub>2</sub>O<sub>2</sub> to a catalase containing medium (Medium-MiR06)'''
+*'''Increase oxygen levels by [[Open_chamber | opening the chamber]]'''
-:Add catalase at a final concentration of 280 IU/mL to the medium at the beginning of an experiment. When oxygen starts to become limited, inject 1-3 µL of an approximately 200 mM H2O2 stock solution. The H<sub>2</sub>O<sub>2</sub> will immediately be degraded to O2; the catalase concentration is high enough to avoid any oxidative stress. In this approach the chamber is not opened and closed, so the disturbance of the system is less and the stabilization phase of the sensor is short compared to a re-oxygenation by opening the chamber.
-*'''Re-oxygenation by opening the chamber'''
-:Lift the stopper and leave a gas volume above the liquid phase (use the stopper-spacer tool to set the stopper in the right position). Leave the chamber open till oxygen is again up to approximately air calibration level. Close the chamber by inserting the stopper completely and wait till the sensor is stable again (may take 5 to 10 minutes).
+:Lift the stopper using the [[Stopper-Spacer | 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 respiratory activity, only little amounts of oxygen are consumed during the stabilization phase of the sensor after closing the chamber.
+: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 ===
-Reach low oxygen levels in a biological experiment may be done by inserting a N<sub>2</sub> bubble into the chamber, waiting until the desired level of oxygen is reached and then destroying the air bubble by closing the stoppers, see how in: [https://wiki.oroboros.at/index.php/Amplex_UltraRed#Nitrogen_injection Nitrogen injection]. However, most often the desired starting value is simply reached by waiting until the sample has consumed the required amount of oxygen and then starting the "oxystat".
+Low oxygen levels can be reached depending on the application. (1) during an experiment with a biological sample this may be performed by injection of N<sub>2</sub> in the gas phase, followed by closing the stoppers once, as described in [https://wiki.oroboros.at/index.php/Amplex_UltraRed#Nitrogen_injection Nitrogen injection]. 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.
-For calibration and instrumental oxygen background purposes we use Dithionite to reduce oxygen levels, however this is not recommended in the presence of biological samples.
+For calibration and for  [[MiPNet14.06 Instrumental O2 background |instrumental O<sub>2</sub> background tests]]  purposes, dithionite is used to reduce oxygen levels, however this is not recommended in the presence of biological samples.