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==MIG-List correspondence==
== Discussion ==
:::: Scientists who have experience with both instruments are invited to extend the discussion:


Von: Mitochondria Interest Group [mailto:MITOCHONDRIA-L@LIST.NIH.GOV] Im Auftrag von Erich Gnaiger. Gesendet: 24 September 2011 23:47
::::Β» [[Microwell respirometry]]
::::Β» [[Talk:MiPNet18.10 O2k versus multiwell|'''Tony Hickey: Calibration is my chief concern''']]
::::Β» [[Talk:Rogers 2011 PLoS One|XFe for isolated mitochondria - a critical discussion]]


An: MITOCHONDRIA-[email protected]
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Betreff: [MITOCHONDRIA-L] Oxygraph-2k for HRR versus XF multi-well method
== MiPNet18.10 O2k versus multiwell ==
Β 
Content of this Email is solely the Responsibility of the Author.Β  Please see Disclaimer at the bottom.--------
Β 
-
Β 
Dear all, dear Pablo:
Β 
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:
Β 
1.1 Instrumental specifications of the XF multi-well system are missing
Β 
1.2 Uncoupled flux does not reflect electron transfer system capacity
Β 
1.3 Lack of validation with high-resolution respirometry
Β 
1.4 Lack of quality control of isolated mitochondria - lessons in
bioenergetics
Β 
* [[Talk:Rogers_2011_PlosOne|Full text of discussion]]
Β 
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".
Β 
(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.
Β 
(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).
Β 
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.
Β 
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_2012_Methods Mol Biol|Pesta and Gnaiger 2012]]).
Β 
Best wishes
Β 
Erich
Β 
DISCLAIMER / CONFLICT OF INTEREST: Dr Erich Gnaiger (Medical University of
Innsbruck, Austria) is Chairman of the Mitochondrial Physiology Society [http://www.mitophysiology.org www.mitophysiology.org].
He is also founder and managing director of OROBOROS INSTRUMENTS [http://www.oroboros.at www.oroboros.at].
Β 
Β 
-----UrsprΓΌngliche Nachricht-----
Von: Mitochondria Interest Group [mailto:[email protected]] Im
Auftrag von Murphy, Anne
Gesendet: 19 September 2011 18:53
Betreff: Re: [MITOCHONDRIA-L] functional mitochondria isolation from primary
cortical cell cultures
Β 
Content of this Email is solely the Responsibility of the Author.Β  Please
see Disclaimer at the bottom.--------
Β 
-
Β 
Dear Pablo,
Β 
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.Β  We did the validation work with the XF24, but it works in
the XF96, as well.Β  Attached is the pdf.
Β 
Best of luck,
Β 
Anne
Β 
Β 
-----UrsprΓΌngliche Nachricht-----
On Sep 18, 2011, at 2:40 PM, Pablo R Castello wrote:
Β 
Content of this Email is solely the Responsibility of the Author. Please see
Disclaimer at the bottom.--------
Β 
- 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
Β 
Β 
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/.
Β 
Β 
== O2k versus multiwell respirometer accessed ==
Β 
[[O2k versus multiwell respirometer]] has been access more than
* 1950 times (2013-01-26) - when the link was added to the front page.

Latest revision as of 08:27, 19 September 2019

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Discussion

Scientists who have experience with both instruments are invited to extend the discussion:
Β» Microwell respirometry
Β» Tony Hickey: Calibration is my chief concern
Β» XFe for isolated mitochondria - a critical discussion

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MiPNet18.10 O2k versus multiwell