Molecular Probes Live/Dead Kit

Nebe-Von-Caron, G g.nebe-von-caron at unipath.com
Fri Jun 18 20:53:42 EST 2004


The combination of CFDA and PI has been used for a long time by numerous
people as cytotoxicity measurement. The lower retention of
carboxyfluorescein resulted in happy highly fluorescent cells whilst the
transition to the PI positive cell was preceded by a lowering in CF
fluorescence. On the other extreme it was possible to use CFDA-SE to
achieve permanently bound green fluorescence to permanently label the
target cells and detect the PI positive cells as permeabilised target
cells. We have also used this combination to investigate the impact of
cell preparation on cell integrity. CF+ cells that remained PI negative
indicated integrity, whilst cells destroyed in the preparation were
positive for both, CF and PI. Cells dead prior to the cell preparation
were CF negative and PI positive.
 
My experiments with Etidium homodimer a long time ago on bacteria showed
the presence of sufficient ethidium to result in dye uptake in cells
that still excluded PI that I did not pursue this dye any further.
However, Mol.Probes claimed that later preps were free of monomer.
Calcein-AM on the other hand has a prolonged retention. Thus I would not
be too surprised to see the move for permeabilisation to go via a double
positive cluster as the influx of one dye might precede the efflux of
the other.
 
Depending on the frequency of your double positives you should be able
to observe them in the fluorescent microscope to identify the
distribution of the two dyes and to exclude aggregation and adhesion of
cell debris to otherwise intact cells. With mammalian cells you should
also be able to exclude the problems of coincidence by means of an
increased light scatter, not so with bacteria, were light scatter goes
for over a log decade. And there particularly with chemoporating agents
I have seen double positives even with CFDA and PI.
 
Regards
 
Gerhard
 
 

	-----Original Message-----
	From: Pyle, Robert H [mailto:Robert.Pyle at STJUDE.ORG] 
	Sent: 15 June 2004 16:37
	To: Cytometry Mailing List
	Subject: Molecular Probes Live/Dead Kit
	
	
	Is anyone using the Molecular Probes Live/Dead,
Viability/Cytotoxicity Kit?  We are trying to put into place a viability
procedure that measures metabolic activity as well as membrane
integrity.  This would replace our usual trypan blue testing.  We have
tried this kit with a FACS Calibur and have problems with dual
expressing cells which Molecular Probes technical assistance has told me
should not exist.  I suspected compensation issues but no amount of
compensation resolves the problem.  If someone out there has any
experience with this kit I would love to ask some questions.
	 
	Thanks,
	 
	R. Haywood Pyle, MS, MT(ASCP), QCYM
	St. Jude Children's Research Hospital
	Immunopathology and Flow Cytometry
	332 North Lauderdale St.
	Memphis, TN  38105
	Tel: 901-495-3399
	Fax: 901-495-2432
	 

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