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

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{{Technical support}}
{{Technical support}}
:::: '''Question:''' Can we set the oxygen concentrations in the O2k to desired levels?


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:
:::: '''Answer: ''' Yes. - It is always possible to increase the oxygen concentration using the combination of catalase in the medium and injections of H<sub>2</sub>O<sub>2</sub>, as described inΒ  [[MiPNet14.13 Medium-MiR06| MiPNet14.3]]. By using the [[TIP2k]], the oxygen concentration 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]].


=== Increasing the oxygen concentration ===
::::Low oxygen levels can be reached depending on the application: (1) On a biological experiment this may be performed by injection of N2 in the gas phase, followed by closing the stoppers once the desired level of oxygen is reached. 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.
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:::: 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.
*'''Increase oxygen levels by adding [[Hydrogen peroxide|H<sub>2</sub>O<sub>2</sub>]] to the [[Catalase|catalase]] containing medium [[MiPNet14.13 Medium-MiR06|MiR06]]'''*
:It is always possible to increase the oxygen concentration by injecting H<sub>2</sub>O<sub>2</sub> into the mitochondrial respiration medium [[MiPNet14.13 Medium-MiR06|MiR06]] (MiR05 plus 280 IU/mL catalase) or [[MiR06Cr]] (MiR06 plus 20 mM creatine) as described in [[MiPNet14.13 Medium-MiR06| MiPNet14.3]]. By using the [[TIP2k]], the oxygen concentrations can be maintained within well defined limits using H<sub>2</sub>O<sub>2</sub> in the [[Microsyringe\500 mm3\TIP2k |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]].
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:When oxygen starts to become limited, inject 1-3 Β΅L of [[Reoxygenation#Preparation_of_200_mM_H2O2_stock_solution|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 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 Β΅M 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.
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:<small>*The mitochondrial respiration medium MiR06 contains catalase. If the respiration medium [[MiR05]] is being used,Β  5 Β΅L of catalase stock (final concentration of 280 IU/mL) can be added toΒ  to allow re-oxygenations with H<sub>2</sub>O<sub>2</sub>.</small>
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* '''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[[Syringe\60 ml\Gas-Injection | 60 mL syringe]] inject oxygen 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.
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*'''Increase oxygen levels by [[Open_chamber | opening the chamber]]'''
: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 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.
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=== 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 (N<sub>2</sub>) into the gas phase, followed by closing the stoppers once the desire oxygen level is reached, as described below:
:* [[Open chamber]]: Before injecting N<sub>2</sub>, the O2k-Chamber needs to be opened to obtain a defined gas volume above the aqueous phase. Β 
:* Using the [[Syringe\60 ml\Gas-Injection | 60 mL syringe]] inject N<sub>2</sub> 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.
:However, often the desired starting value is simply reached by waiting until the sample has consumed the required amount of oxygen.
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:To perform [[Zero calibration|zero calibration]] oxygen should be removed by the addition of [[Dithionite|dithionite]] or biological sample.
:To reduce the oxygen levels during an [[MiPNet14.06 Instrumental O2 background |instrumental O<sub>2</sub> background test]], [[Dithionite|dithionite]] should be used.

Latest revision as of 09:21, 17 June 2020


                  


O2k-Open Support

Talk:Setting the oxygen concentration



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Question: Can we set the oxygen concentrations in the O2k to desired levels?
Answer: Yes. - It is always possible to increase the oxygen concentration using the combination of catalase in the medium and injections of H2O2, as described in MiPNet14.3. 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 TIP2k microsyringes. Such specification is called "oxystat" approach and supply appropriate templates for controlling the TIP2k. See also MiPNet12.10.
Low oxygen levels can be reached depending on the application: (1) On a biological experiment this may be performed by injection of N2 in the gas phase, followed by closing the stoppers once the desired level of oxygen is reached. 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.