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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).
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@pasteur.fr _/ _/ _/
Tel: 33 (01) 40 61 31 29 _/_/_/ _/ _/
Fax: 33 (01) 45 68 86 39 INSTITUT PASTEUR PARIS
______________________________________________________________
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