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Talk:O2k-Specifications

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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

Content of this Email is solely the Responsibility of the Author. Please see Disclaimer at the bottom.--------

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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


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_2010_Protocols|Pesta and Gnaiger 2011).

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.



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

Content of this Email is solely the Responsibility of the Author. Please see Disclaimer at the bottom.--------

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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



Ursprüngliche Nachricht-----

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

Content of this Email is solely the Responsibility of the Author. Please see Disclaimer at the bottom.--------

- 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


Medical advice and information that could identify an individual is discouraged on this list. List members are reminded of their responsibility to critically evaluate the content of the postings. The information, opinions, data, and statements contained herein are not necessarily those of the U. S. Government, the National Institutes of Health (NIH), or the Mitochondria Interest Group (MIG) and should not be interpreted, acted on or represented as such. Please see the MIG website: http://sigs.nih.gov/mito/.