Re: Fluorochromes choice

From: William Telford (TelfordW@mail.nih.gov)
Date: Sun Nov 03 2002 - 03:24:21 EST 




Hello Michal...


Although I agree with almost all the comments so far about doing 6-color
on the original LSR, you still might want to give the UV-excited
fluorochrome AMCA (or its more recent descendant Alexa Fluor 350) a try
as your sixth color.  AMCA is actually a reasonably bright
fluorochrome - unfortunately its emission at ~450 nm falls in an area of
high cellular autofluorescence, making it appear dim relative to
background.  However, if you use it to label a densely expressed
marker, it might be bright enough to visualize.  We've made it work
in the past for a few highly expressed surface markers, such as CD8 on
mouse T cells - expect at least a one-to two log decrease in fluorescence
compared to FITC.  Use your 424/44 filter for detection.  We
have also tried AMCA and Alexa Fluor 350 with a variety of UV lasers -
although its emission peak is around 350 nm, the HeCad 325 nm line still
seems to excite it as well as (or no worse than, anyway) the longer argon
and krypton UV lines.  You can use Alexa Fluor 350-streptavidin to
label a biotin-conjugated antibody, or use the Zenon kit from MPI to make
a "conjugate".  


 From an instrument standpoint, you might want to get the 4-2-2 LSR
upgrade to do four colors off the 488 nm and two off the 633 nm.  I
know this configuration exists for the LSR, although I'm not absolutely
sure if BD is routinely doing upgrades to it.  Alternately, you
might want to consider investing in the 405 nm violet laser diode upgrade
and use Cascade or Pacific Blue as your sixth colors, which give much
better signal-to-noise ratios than AMCA.


Enjoy,


Bill Telford

NCI-NIH


(and yes, no financial stake in Molecular Probes, BD Bioscience, or
anyone else for that matter!)


At 11:21 AM 10/30/2002 -0800, Michal Abel wrote:



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 ?



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















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