RE: mitochondrial mass measurement ***Commercial Response***

Hi Derek and David,
 
We did a quick feasibility test of Invitrogen's Organelle Lights
mitochondrial GFP probe versus TMRE in the same Colo205 cells using the
ImageStream. We found very good co-localization of the signals. The GFP
signal was seemingly independent of mitochondrial potential and
exhibited much lower background staining than TMRE. I think it would be
a good indicator of mitochondrial mass in the cell types that work with
the Organelle Lights transfection system. 
 
Best,
David
 
David Basiji, Ph.D.
President and CEO, Amnis Corporation
2505 Third Ave., Suite 210
Seattle, WA 98121
+1 206 374 7165 direct
+1 206 919 3342 mobile
+1 206 576 6895 fax
 
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________________________________

	From: Derek Davies [mailto:derek.davies@cancer.org.uk] 
	Sent: Monday, January 07, 2008 1:24 PM
	To: Cytometry Mailing List
	Subject: Re: mitochondrial mass measurement
	
	
	Hi David,
	
	I don't 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 ('live', 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@gsk.com"
<David.C.McFarland@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
		

	
	
	-- 
	***************************************************************
	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@cancer.org.uk
	Web Page: http://science.cancerresearchuk.org/sci/facs/
	
	In tenebris lux    
	***************************************************************