mitochondrial mass measurement ***Commercial Response***

Jayaraman, Sundararajan anue2468 at uic.edu
Wed Jan 9 21:50:46 EST 2008


I compared three mitochondria-specific dyes: TMRE, H2-CMX-Ros and
MitoTracker Red580.  TMRE is highly membrane potential dependent.
H2CMX-Ros is variable while MTR 580 is not at all dependent on membrane
potential.  The data can be found in the following reference:

Jayaraman S. Flow cytometric determination of mitochondrial membrane
potential changes during apoptosis of T lymphocytes and pancreatic beta
cells. Comparison of tetramethylrhodamineethylester (TMRE),
chloromethyl-X-rosamine (H2-CMX-Ros) and MitoTracker Red580 (MTR580). J
Immunol Methods 2005;306:68-79.

SJ

S. Jayaraman, Ph.D.
Associate Professor of Surgery
University of Illinois at Chicago
909 South Wolcott Avenue-704E MSB-M/C 790
Chicago, IL 60612
Phone: 312-355-5133
Fax: 312-355-1497


On Tue, January 8, 2008 2:54 pm, David Basiji wrote:
> 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
>
> This email and any attachment contain information which is intended for
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>
>
> ________________________________
>
>	From: Derek Davies [mailto:derek.davies at 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 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
>
>
>
>
>	--
>	***************************************************************
>	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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