FW: Measuring mitochondrial/metabolic activity by flow

Darzynkiewicz, Zbigniew Z_DARZYNKIEWICZ at nymc.edu
Thu Jan 18 16:49:59 EST 2007


Ooops, sorry, the proper webpage for this article is:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dop
t=citation&list_uids=17227291&query_hl=1&itool=pubmed_docsum




-----Original Message-----
From: Darzynkiewicz, Zbigniew 
Sent: Thursday, January 18, 2007 4:46 PM
To: cyto-inbox
Subject: RE: Measuring mitochondrial/metabolic activity by flow

The mitochondrial potential probes do measure just that: mitochondrial
potential (and if the fluorescence is integrated over the whole cell
they also reflect the number/mass of mitochondria per cell). But
mitochondrial potential may not be directly proportional to the actual
metabolic activity. Needless to say, the ratiometric probes such as JC1
are not sensitive to the number/mass of mitochondria.
 Because the by-product of metabolic activity is generation of oxidative
radicals I suggest the use one of the probes sensitive to oxidation
(e.g. dihydrorhodamine or carboxyl-dichlorodihydrofluorescein diacetate
(H2DCF-DA). We have recently used H2DCF-DA to compare metabolic activity
of stimulated vs nonstimulated lymphocytes and relate it to constitutive
DNA damage: See the abstract in PubMed:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Pager&DB=pubmed


Zbigniew Darzynkiewicz, M.D., Ph.D.
Professor of Pathology and Medicine
Director, Brander Cancer Research Institute
New York Medical College
BSB, Room 438
Valhalla, N.Y. 10595

www.darzynkiewicz.com/zbigniew/


-----Original Message-----
From: Rosemary Clarke [mailto:r.z.clarke at dundee.ac.uk] 
Sent: Thursday, January 18, 2007 6:27 AM
To: cyto-inbox
Subject: Measuring mitochondrial/metabolic activity by flow

Dear All,

I hope that someone out there may be able to help. Has anyone every  
used mitochondrial membrane potential dyes (such as JC1) to monitor  
changes in cell metabolic status? This has been suggested as an  
additional experiment by a reviewer for a recently submitted paper.  
However, I have never seen this sort of dye used in this way, only as  
a marker for the loss of membrane potential during apoptosis. I fail  
to see how it would be possible to distinguish a cell entering	
apoptosis from one that has low metabolic activity anyway. Initial  
attempts to look for differences between highly metabolically active  
cells and metabolically inactive cells have not shown any  
differences, although the dye is working in control samples (ie live,  
dying or dead cells). Any comments or suggestions of alternative  
assays would be much appreciated. We would do radioactive amino acid  
or glucose uptake assays in the traditional way but we are limited by  
cell number (thousands as opposed to millions), hence our need to use  
flow cytometry.

Thanks in advance for your help.

Rosie

Dr Rosie Clarke
Division of Cell Biology and Immunology
WTB/MSI
University of Dundee
Dundee
Scotland
UK
Tel: (01382) 385780
E-mail: r.z.clarke at dundee.ac.uk






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