Talk:Chemical background correction of oxygen flux

Customer question:

We have been running ascorbate TMPD protocols at the end of some experiments on permeabilised fibres and need to apply the chemical correction in addition to the instrument background correction. We have added Cyt C then ascorbate then TMPD and allowed the O2 conc to decay to zero. We then select a series of marks on the linear part of the traces and use those numbers in the chem. back. template to generate values for slope and intercept. What we are not sure is where to use the R0 value (when chamber is anoxic) and exactly where we copy in the slope and intercept values. Do they simply go into the O2 correction to add R0 and then edit experiment menu to put in a’ & b’ values that are the sum of chemical and chamber backgrounds?

Answer:

• Additivity of background parameters: I think the confusion is due top a inconsistency between MiPNet10.04. If you choose a background corrected flux display (as demonstrated in MiPNet10.04 ) for determining the chemical background, the result (a') has to be added to a0 to get the final a0', as explained in MiPNet10.04, page 11. The parameters a0', b0' are then used in the "edit experiment file. However, if you used a "O2 slope uncorrected" plot to export the data from, as done in the example in the spreadsheet template, you naturally will already get the combined parameters that can be used in th edit experiment window. So it depends on which plot you selected before exporting the data to the spreadsheet program. Sorry for the confusion!

• Inclusion of R0: In MiPNet 10.04 it is suggested only to include the values down to about 40 to 50 µM, because only in this range there is a linear relationship. R0 is only used for the zero calibration of the oxygen sensor not for determining the chemical background parameters

Please note that in any case these new background parameters should only be applied for those phases of the experiment in which the O2 consuming chemicals are actually present!

best greetings

Mario

Fasching Mario 14:45, 17 October 2011 (CEST)

Customer Question: After a long period, where I have not measured COX, I am about to start a new experiment. The way I have use before is the method described in MipNet 10.04. But now I have read Erich and Dominiks “OXPHOS Protocols for human cells and Permeabilized fibers from small biopsies of human muscle” and here they describe a method where they add KCN og Azide to get ROX and as I read it , they after that subtract the autoauxidation from the chemical background found like in mipNet 10.04.. Then I have read MipNet 06.06 and that is also a different way. Can you point out exactly how to perform an chemical background.?

Do I make a chemical background as in mipNet 10.04 and then subtract that (autooxidation) from the COX respiration and from the ROX when COX is inhibited with KCN or Azide? Or what

I am a bit confused – hope you can help me --Fasching Mario 09:40, 19 January 2012 (CET)