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MiPNet19.03 O2k-cleaning and ISS

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O2k-Chamber cleaning SOP and Integrated Suction System (ISS). »Bioblast pdf«

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OROBOROS (2014-04-14) Mitochondr Physiol Network

Abstract: Fleischmann S, Gnaiger E (2014) O2k-Chamber cleaning SOP and Integrated Suction System (ISS). Mitochondr Physiol Network 19.03(01):1-4.

>> Product: O2k, O2k-Catalogue


O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Instruments;methods 





HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

O2k-SOP 

Supplement

A properly assembled chamber can remain in the oxygraph for a long time. However to avoid the chamber getting stuck due to dried media and to detect damages or contaminations we recommend to disassemble, clean and reassemble the chamber at least every 1 - 2 years.

General cleaning instructions

There are at least three fundamentally different kind of contaminations that can accumulate in the measuring chamber of the oxygraph and cause problems. All of them have to be treated in different ways:

  • Biological contamination: The ideal counter agent is 70% Ethanol with 30% water (NOT 100% Ethanol). If this does not help the biological contamination may be embedded in e.g. protein contamination and the glass chamber has to be disassembled and cleaned as described below:
  • Protein contamination and other macroscopic deposits. After long use a whitish deposit can form at the glass walls of the chamber. Additionally, small glass splinters (difficult to see) may become embedded in such a deposit. This will be detected by a jumping stirring bar or a stirring bar getting stuck. In this case the glass chamber should be removed from the oxygraph and treated with concentrated hydrochloric acid at least over night. Use a stirring plate and the O2k stirring bar, in this way the stirring bar is cleaned simultaneously.
  • Contamination by hydrophobic inhibitors: The ideal counter agent for this case is pure ethanol.


Further information >>


Cleaning the O2k-Chamber between experiments

For routine use of the O2k with the glass chambers inserted in the respirometer, cleaning should be done as explained in detail.

Clean the chamber after an experiment involving lipid-soluble inhibitors (such as oligomycin, rotenone, or antimycin A):

  • The chamber must be cleaned rigorously with water (washing water-soluble inhibitors such as azide), and ethanol (100%), since lipid-soluble inhibitor(s) are difficult to be washed out from the chamber and may inhibit mitochondrial respiration in subsequent experiments.
  1. Siphon off the cell/mitochondrial suspension at the end of the experiment and rinse the chamber with distilled water 5 times, by filling the chamber up to the rim. During all washing steps of the chamber, stirring has to be switched on.
  2. Rinse the surface and capillary of the stopper with distilled water several times properly.
  3. Fill the water-cleaned chamber with 70 % ethanol and replace the stopper making sure that the ethanol fills up the receptacle, and leave for 5 min.
  4. Remove the stopper and siphon off the ethanol to empty the chamber. Repeat these cleaning steps with 70 % ethanol three times.
  5. Then fill the chamber with absolute ethanol (99.6 %) and insert the stopper making sure that the ethanol fills up the receptacle. Place the perspex cover on top of the stopper and leave for 15-20 min.
  • If you are finished with experiments for the day, replace absolute ethanol with 70% ethanol for storage.
  • If you will start a new experiment, rinse chamber with distilled water 5 times, and rinse the surface and capillary of the stopper with distilled water several times properly.

Rinse the stopper by holding it at the receptacle, not at the shaft that fits into the chamber to avoid contamination.

  • After measurements on permeabilized fibers it is advisable to remove the stirrer from the chamber and to clean it mechanically, as fibers may be very sticky and may not be removed by the standard celaning procedure.

Cleaning with "dead cells":

  • Every once in a while it might be necessary to wash the chambers with "dead cells" obtained from a e.g. former cell-experiment (frozen at -20°C). Inhibitors will be taken up by the 'dead cells' when filling the chamber up to the rim with a 'dead cell' suspension. The suspension should be left in the chambers with inserted stoppers for at least 30 min.


Unintentional introduction of inhibitors to the O2k-Chamber

Inhibitors may also be introduced unintentionally, one example being 70% ethanol used in hospital settings containing antiseptics. Such inhibitors may accumulate e.g. in plastic parts and inhibit subsequent experiments. See the discussion page.

Further details

>> O2k-Chamber
>> MiPNet12.08 O2k-Calibration
>> MiPNet14.06 InstrumentalBackground