mitochondrial mass measurement

Derek Davies derek.davies at cancer.org.uk
Mon Jan 7 16:24:16 EST 2008


Hi David,

I don¹t really have an answer for you but would certainly be interested in
any replies.

We have used NAO to monitor apoptosis (indirect release of cytochrome c
after peroxidation of cardiolipin) - in most situations it works well but we
have also seen situations where healthy uniform cells show a (Œlive¹, PI
negative) population of cells that would appear to be non-apoptotic. I think
you have to be very careful with dye concentration and the optimal may vary
a lot between cells ­ it is also an extremely bright dye that can be
difficult to combine with other fluorochromes. As you say, there are a lot
of conflicting reports in the literature as to action of the dye and the
interpretation of the staining.

Way back, with Gerard Evan, I did look at the early versions of the
MitoTracker dyes, I think we used the Green and Orange versions and they did
seem to work well in that they showed the expected changes ­ now though I am
not sure that we were quite looking at it in the right way. Comparison of
the dyes in an experimental situation would be good if anyone has the time
to do that! Most of this was done in suspension but the LSC might be a good
approach so you can see what the cells look like as well.

Derek


On 4/1/08 5:35 pm, "David.C.McFarland at gsk.com" <David.C.McFarland at gsk.com>
wrote:

> 
> Once upon a time I would have said nonyl acridine orange (NAO) labelling of
> cardiolipin was the method of choice for measuring mitochondrial mass by flow.
> But over the years, a few articles have been published that cast doubt on the
> validity of this assay.  First, some researchers have shown that NAO staining
> is mitochondrial membrane potential (MMP) dependent.	This is problematic,
> especially if the point is to show changes in mass irrespective of MMP or if
> fixed cells are to be used.  And secondly, at least one group has demonstrated
> NAO is not specific for cardiolipin since it very well labels
> cardilipin-defiicient cells.	So, my question is, what do you recommend for
> flow- or LSC-based measurement of mitochondrial mass?  Can anyone comment on
> the MitoTracker or MitoFluor series of dyes (or a specific dye in the series)
> from Invitrogen-Molecular Probes?  I would be very interested to know if
> someone has compared all the MitoTracker/Flour dyes head-to-head.
> 
> Thanks in advance for your replies,
> 
> Dave 
> 
> References of interest:
> 
> Gohil, et al.  Analytical Biochemistry343 (2005)350-352.
> Keij, et al.	Cytometry 39 (2000) 203-210.
> 
> 
> David McFarland
> Principal Scientist
> GlaxoSmithKline


-- 
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Derek Davies, FACS Laboratory, London Research Institute,
Cancer Research UK, 44 Lincolns Inn Fields, London, UK.

Tel: (44) 20 7269 3394
FAX: (44) 20 7269 3479
mobile: 07790 604112
e_mail: derek.davies at cancer.org.uk
Web Page: http://science.cancerresearchuk.org/sci/facs/

In tenebris lux    
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