Hi,
When studying the respiratory function of isolated mito. by measuring OCR using the Seahorse-
I'm using a buffer (MAS) that contains pyruvate+ malate.
After measuring the basal respiration- ADP, Oligomycin, FCCP and sodium azide are being added by sequential injections:
Basal respiration--> ADP injection--> Oligomycin injection--> FCCP injection--> sodium azide injection.
I'm getting the expected trends, but I was wondering what could be the reason for getting lower OCR after adding FCCP (in comparison to the OCR after adding ADP)?
I saw in many different papers (where similar injections were done) that sometimes the OCR is higher after adding FCCP, but sometimes the OCR is lower after adding FCCP (again- in comparison to the OCR after adding ADP).
Could you help me to figure it out.....? I'm sure that I'm missing something :)
Many thanks for your help!
Dear Chen Lesnik,
Seahorse is one of the instruments with too many inborn sources of errors.
Too long to explain. The sequential addition of inhibitors is one of the most stupid approaches to study mitochondria. It gives no valuable information, except showing that the user does not know the basics of mitochondrial functions. I am rough because too many young researchers stick to this method. Use any real polarography, except Oroboros, which is just waste of money.
You should also know that pyruvate with or without malate interacts nonezymatically with hydrogen peroxide and therefore the concentration of pyruvate changes very quickly. One of the disadvantages of the Seahorse is that the relatively high surface and small volume of the medium allows fast interaction with ozone in the air, which destroys pyruvate. In addition you check the amount H2O2 in you medium. Finally, FCCP much more often inhibits respiration rather than stimulate. Find and read my book Practical mitochondriology for explanation.
My guess is concentration of FCCP is not optimal (most likely too high). High FCCP suppress respiration. I would run small-scale FCCP titration for your exact conditions (buffer, mitochondrial concentration etc) to find it. In isolated mitochondria, uncoupling concentration of FCCP is much lower than for cell suspension.
BTW, if you have relatively pure mitochondria you do not have to add azide. Instead, you may think about using rotenone+succinate mix injection.
Thanks Alexander V Panov for your notes, and thanks Sergei Baranov!
The FCCP conc. was optimized.
After consulting with Agilent, I understood that it's because I use isolated mito. (and not intact cells).
Thanks!
Chen
Yes, Seahorse provides a very narrow and restricted snapshot of mitochondrial function. When using the Seahorse protocol subsequent injections of ADP, Oligomycin, and FCCP, many factors and conditions affect the FCCP response in isolated mitochondria. Substrates such as Pyruvate, Malate, Glutamate, Succinate etc. all give different FCCP responses compared with ADP stimulated OCR. Some times higher, sometimes lower.
Most importantly, the response is variable between tissue types. Liver and Skeletal muscle isolated mitochondria often do have increased OCR at the FCCP step. Kidney mitochondria I have optimised always had lower OCR at the FCCP step (with Glu/Mal). I have seen that Heart is also often lower.
When comparing isolated mitochondria to intact cells the FCCP response can be the opposites of each other. I think we expect the isolated mitochondria to do the same as cells in seahorse assays because Liver tissue is commonly used and it does have a reserve capacity (increased with FCCP) in both cells and isolated mitochondria. I do not think isolated mitochondria give much useful information on reserve capacity, and they do not reflect the actual reserve capacity available to a whole cell.
Chen Lesnik, can you tell what concentration of FCCP you used for isolated mitochondria. I am also getting the decreased OCR after adding the FCCP (0.5UM) with isolated mitochondria.
Abhishek Kumar Singh You can also titrate ADP levels and use DNP instead of FCCP. DNP gives very consisting results.
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