Talk:MiPNet18.10 O2k-Specifications

Bioenergetics made simple (Erich Gnaiger)

Misleading simple interpretations: Coupling control in intact cells can be simply studied in a sequence of ROUTINE respiration (R), oligomycin-induced LEAK respiration (L), and evaluation of Electron transfer system capacity (E) (Hütter et al 2004). The R/E flux control ratio provides information on ROUTINE respiratory activity relative to ETS capacity (Gnaiger 2008, Gnaiger 2009). In commercially oriented (XFe) discussions, David Nicholls interprets the equivalent of the R/E flux control ratio as an index of reserve capacity. This is not supported by modern concepts on coupling control and is refuted by experimental evidence in human cell models and many tissue preparations (Gnaiger 2012 MitoPathways). This unjustified simplification (Bioenergetics made simple) ignores along the lines of outdated terminology (State 3 and State 3u) the fact that OXPHOS capacity (P) cannot in general be determined by noncoupled states of respiration (E). But E needs to be corrected for the P/E ratio if any apparent reserve capacity of ROUTINE respiration should be determined. This may still be considered as a very simple concept, but apparently not suffiently simple for XFe advertising.

MIG-List correspondence

Von: Mitochondria Interest Group [1] Im Auftrag von Erich Gnaiger. Gesendet: 24 September 2011 23:47

An: [email protected]

Betreff: [MITOCHONDRIA-L] Oxygraph-2k for HRR versus XF multi-well method

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Dear all, dear Pablo:

In a discussion on high-resolution respirometry in relation to the publication by Rogers et al 2011, I raised the question: "High throughput without high output?" with the following topics:

1.1 Instrumental specifications of the XF multi-well system are missing

1.2 Uncoupled flux does not reflect electron transfer system capacity

1.3 Lack of validation with high-resolution respirometry

1.4 Lack of quality control of isolated mitochondria - lessons in bioenergetics

• Full text of discussion

I would be glad to receive your scientific input to this discussion, which has been triggered by two messages circulated to the MIG list under the heading "functional mitochondria isolation from primary cortical cell cultures".

(1) Dr. Anne Murphy attached a pdf-reprint that does not deal with mt-isolation procedures from cell cultures but describes the 'validation' of the XF multi-well method for isolated mitochondria.

(2) The comment by Prof. David Nicholls: "Look to see whether a colleague has a Seahorse" (good science practice would require a disclaimer statement to be added, in line with good practice on the MIG list).

The original question raised by Dr. Pablo R. Castello ("functional mitochondria isolation from primary cortical cell cultures") stimulated a useful discussion, even if the subject changed from 'mt-isolation procedure' to the platform of measurement. I would like to learn more from experiences comparing the Oxygraph-2k for HRR and the XF multi-well method.

Following the suggestion by Prof. Lech Wojtczak, I would also recommend to use a validated method for permeabilization of cells with fully functional mitochondria, and application of advanced diagnostic protocols with multiple substrate-uncoupler-inhibitor titrations (Pesta and Gnaiger 2012).

Best wishes

Erich

DISCLAIMER / CONFLICT OF INTEREST: Dr Erich Gnaiger (Medical University of Innsbruck, Austria) is Chairman of the Mitochondrial Physiology Society www.mitophysiology.org. He is also founder and managing director of OROBOROS INSTRUMENTS www.oroboros.at.

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Ursprüngliche Nachricht-----

Von: Mitochondria Interest Group [2] Im Auftrag von Murphy, Anne Gesendet: 19 September 2011 18:53 An: [email protected] Betreff: Re: [MITOCHONDRIA-L] functional mitochondria isolation from primary cortical cell cultures

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Dear Pablo,

Since you have a Seahorse, you may be interested in making the small yield of mitochondria from the cultured neurons go a lot further experimentally. We recently published a paper on methodology for using isolated mitochondria in the Seahorse. We did the validation work with the XF24, but it works in the XF96, as well. Attached is the pdf.

Best of luck,

Anne

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Ursprüngliche Nachricht-----

On Sep 18, 2011, at 2:40 PM, Pablo R Castello wrote:

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- Dear David, Anne, Gloria, Tibor and Charles, Thank you so much for you advice. David, I already ran almost 80 plates on both a XF 24 and a XF 96 only this year using primary cortex cell cutures. I found a very interesting model of mitochondria damage that is allowing us to test potential neuroprotective small molecules. The idea is to validate my findings using a different analytical method. We have a polarographic instrument, and I thought that It would be a good idea to isolate mitochondria from our stressed cultures and analyze them with this method. From the advice of you all, It seems difficult (because the high number of cells) but not impossible. What do you think? I strongly appreciate this discussion. Best Regards, Pablo

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