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

Latest revision as of 10:21, 17 June 2020

Talk:Setting the oxygen concentration

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

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.

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 {{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 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.
+:::: '''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 in  [[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.
-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]].
+:::: 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.
-An experiment needs not necessarily be terminated, because of running out of oxygen. There are different possibilities to re-oxygenate.
-* '''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.
-*'''Re-oxygenation by adding H<sub>2</sub>O<sub>2</sub> to a catalase containing medium (Medium-MiR06)'''
-: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).
-: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 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.
-=== 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".
-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.