I agree that I haven't seen anything useful for immunophenotyping using
the 325 nm line, although I do have a couple more that I will try in the
near future. I know that some people have said that they can make Alexa
350 work, but it didn't work very well in my hands. However, the 325 nm
line IS useful for exciting aminostilbamidine methanesulfonate. We use it
frequently for dead cell discrimination. It gives resolution between live
and dead better than 7AAD and almost as good as PI. It is available from
Molecular Probes.
If you're interested in getting 2 colors from the UV/V range (besides
Indo) see if you can swap your 325 nm for a 405 nm. This would allow you
to use Cascade Blue and Yellow. Talk to your sales rep to see what you
can work out.
One more tip: If you start to use more of these "exotic" fluors, you'll
be hard pressed to find them conjugated to your Abs of choice. Another
reagent from Molecular Probes that has really opened some doors for us is
their Zenon Ab conjugation kit. They have a wide range of fluors
available. They have kits to conjugate mouse IgG1 and rabbit Ig and I
believe they will also have kits for mouse IgG2a and 2b in the near
future. It's good stuff!
Happy flowing,
Dave
David McFarland
GlaxoSmithKline
----- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 04-Nov-2002 09:52
-----
"Marty Bigos" <mbigos@gladstone.ucsf.edu>
31-Oct-2002 11:34
To: "Cytometry Mailing List"
cc:
Subject: Re: Fluorochromes choice
Dear experts in flow cytometry,
I have a possibility to use 6 colors FACS. The trouble is that I am not
sure it is possible with the filters I have.
I would very much appreciate any help.
The instrument has three lasers: 325, 488 and 633 nm and I can use
following filters:
380/LP
400/40
424/44
500/11
510/20
530/28
575/15
610/20
660/13
670/LP
682/13
Is it possible to find 6 different flourochromes, which would be
efficiently differentiated with these filters/lasers ?
There are no useful fluorochromes for phenotyping that are excited by 325
nm. So you are limited to the 488 and 633 excitation.
In addition to bandpass detection filters, it is also important to
optimize the beamsplitters and the detectors, especially for measuring the
red emitting dyes.
Six dyes that would be useful for your system are:
Using 488 nm excitation:
FITC
PE
PE-TR (aka ECD from Beckman-Coulter)
PE-Cy7
and using the 633 nm excitation:
APC
APC-Cy7
We use these regularly on our Vantage SE for 6 color work.
However, without knowing your instrument configuration I could not say
whether they would work for you. I would guess that from the filters you
described above, you would have trouble detecting PE-Cy7, APC-Cy7, and
quite possibly APC.
Marty
Thank you very much for your help.
Michal
--
Michal Abel, MD, PhD
Laboratory of Clinical Immunology
INSERM U25
Necker Hospital, 161 Rue de Sèvres. Paris
tel: 33 1 42 19 28 87
fax: 33 1 44 49 53 74
--
Marty Bigos
Director, Flow Cytometry Core Laboratory
Gladstone Institute of Virology and Immunology
Mail:
Box 419100
San Francisco, CA 94141
Packages:
365 Vermont Street
San Francisco, CA 94103
Direct Delivery (FedEX, UPS):
Gladstone Institute
San Francisco General Hospital
Building 3 Rm 509
1001 Potrero Avenue
San Francisco, CA 94110
(tel) 415-695-3832
(fax) 415-826-8449
mbigos@gladstone.ucsf.edu
http://gladstone.ucsf.edu
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