Dead Cell Marker on 633 nm FL4 ?

tom_frey@bdis.com tom_frey at bdis.com
Thu Nov 7 22:08:25 EST 1996



In response to Matthias Haury's query (copied below) I have used TO-PRO-3 as  
live dead discriminator with red excitation.  Note that the -3 is important, 
Molecular Probes also sells TO-PRO-1!

For TP3, saturating concentration is 2-4 uM.  I routinely use 10-40 nM for live 
vs dead discrimination.  Since this will require creating a diluted stock, do 
the dilution in DMSO or else do not store an aqueous (PBS) dilution in 
polystyrene - the dye concentration will decrease over time possibly due to loss 
to the tube wall.  Polypropylene is better.

As I just remove the deads, I have not been too careful about checking the 
compensation.  There is some blue excitation (FL3 emission) of TO-PRO-3 that is 
a problem at saturation.  I don't think it will be a difficulty in this 
application, and you may not need any compensation.

You could also consider 7-AAD, which is fairly well confined to FL3.  You may 
have experience with the small amount of FL2 emission it produces.  It appears 
to me to have virtually no FL4 on the Calibur and you could probably use FITC, 
PE and APC with minimal interference also.  The smaller shift it produces would 
make it harder to go for 4-color + live/dead which you might be able to get away 
with by optimizing TO-PRO-3 (Matthias' other post suggests he tries this with 
PI).

Tom_Frey at bdis.com

Subject: Dead Cell Marker on 633 nm FL4 ?

Those guys out there that have already experience with the FACScalibur 4
Colors (or equivalent setup on another machine), is there a dye that you
can use on the FL4 to exclude dead cells, while still using FITC, PE,
Tricolor for Surface markers ?

I remember to have heard something about TOPRO etc, but which works the best ?

Thanks for details (dye, manufacturer, compensations, staining concentrations).

Merci,

Matthias

______________________________________________________________
Matthias Haury                      _/_/_/    _/_/_/    _/_/_/
Flowcytometry - Immunology           _/     _/    _/  _/    _/
Institut Pasteur Paris              _/     _/_/_/    _/_/_/
Email: mhaury at pasteur.fr           _/     _/        _/
Tel:   33 (01) 40 61 31 29      _/_/_/   _/        _/
Fax:   33 (01) 45 68 86 39      INSTITUT PASTEUR   PARIS
______________________________________________________________





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