Contamination by hydrophobic inhibitors

Problem

Lasting effect of inhibitors (i.e. rotenone, antimycin A), as observed by persistent low fluxes, when working with permeabilized fibers.

Cleaning the Chamber between experiments

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.
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 30 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.

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.