Contamination by hydrophobic inhibitors: Difference between revisions

Revision as of 16:28, 13 September 2010

Problem

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

Cleaning the Chamber

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

The chamber must be cleaned rigorously with ethanol (100%), since such inhibitor(s) are difficult to be washed out from the chamber and may inhibit mitochondrial respiration in subsequent experiments.
Siphon off the cell/mitochondrial suspension at the end of the experiment and rinse the chamber with distilled water three times, by filling the chamber up to the rim.
Also rinse the surface and capillary of the stopper with distilled water properly.
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.
Remove the stopper and siphon off the ethanol to empty the chamber. Repeat these cleaning steps with 70 % ethanol three times.
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.
At the end of this rigorous cleaning procedure remove the stopper and place it inverted (with the receptacle at the bottom) on a clean paper towel.
Siphon off the ethanol from the chamber and rinse it with distilled water three times.
Rinse the stopper by holding it at the receptacle, not at the shaft that fits into the chamber to avoid contamination.
After the cleaning procedure store the chamber in 70% ethanol.

Cleaning with "Dead Cells"

Template:Technical service

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-'''Question:''' When working with permeabilized fibers from mouse heart, we had serious problems to wash out inhibitors (rotenone, antimycin A), even after careful washing of the chambers with pure ethanol (see below). In some chambers, the inhibitors exerted a lasting effect, as observed by persistent low fluxes in these particular chambers. In most cases, fluxes returned to normal values after titration of ascorbate and TMPD, suggesting a persistent inhibition upstream of cytochrome c oxidase.
+==Problem==
-The effect could be removed only after disassembling the chambers, and immersing chambers, stirrers, stoppers in a beaker with ethanol, and one time even that did not work immediately. We have never observed such a serious inhibitor problem when working with cultured cells or isolated mitochondria, nor when working with permeabilized rat cardiac fibers or permeabilized liver tissue. And we have no idea for the reason that this observation was made only in a series of experiments with permeabilized mouse cardiac fibers.
+Lasting effect of inhibitors (i.e. rotenone, antimycin A), as observed by persistent low fluxes, when working with permeabilized fibers.
+==Cleaning the Chamber==
+'''Clean the chamber after an experiment involving lipid-soluble inhibitors (such as oligomycin, rotenone, or antimycin A):'''
+*The chamber must be cleaned rigorously with ethanol (100%), since such inhibitor(s) are difficult to be washed out from the chamber and may inhibit mitochondrial respiration in subsequent experiments.
+*Siphon off the cell/mitochondrial suspension at the end of the experiment and rinse the chamber with distilled water three times, by filling the chamber up to the rim.
+*Also rinse the surface and capillary of the stopper with distilled water properly.
+*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.
+*Remove the stopper and siphon off the ethanol to empty the chamber. Repeat these cleaning steps with 70 % ethanol three times.
+*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.
+*At the end of this rigorous cleaning procedure remove the stopper and place it inverted (with the receptacle at the bottom) on a clean paper towel.
+*Siphon off the ethanol from the chamber and rinse it with distilled water three times.
+*Rinse the stopper by holding it at the receptacle, not at the shaft that fits into the chamber to avoid contamination.
+*After the cleaning procedure store the chamber in 70% ethanol.
-'''Solution:''' Cleaning the Chamber
-Clean the chamber after an experiment involving lipid-soluble inhibitors (such as oligomycin, rotenone, or antimycin A): The chamber must be cleaned rigorously with ethanol, since such inhibitor(s) are difficult to be washed out from the chamber and may inhibit mitochondrial respiration in subsequent experiments. Siphon off the cell/mitochondrial suspension at the end of the experiment and rinse the chamber with distilled water three times, by filling the chamber up to the rim. Also rinse the surface and cannula of the stopper with distilled water properly. 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. Remove the stopper and siphon off the ethanol to empty the chamber. Repeat these cleaning steps with 70% ethanol three times. Then fill the chamber with absolute ethanol (99.6%) and insert the stopper making sure that the ethanol fills up the receptacle. Replace the perspex cover on top of the stopper and leave for 15-20 min. At the end of this rigorous cleaning procedure remove the stopper and place it inverted (with the receptacle at the bottom) on a clean paper towel. Siphon off the ethanol from the chamber and rinse it with distilled water three times. Rinse the stopper by holding it at the receptacle, not at the shaft that fits into the chamber to avoid contamination. After the cleaning procedure store the chamber in 70% ethanol.
 Cleaning with "Dead Cells"
-{{#set: Technical service = chamber |Technical service = inhibitor | Technical service = contamination}}
+{{#set: Technical service = chamber |Technical service = Inhibitor | Technical service = contamination | Scientific service= Inhibitor }}
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 {{Technical service}}