Re: Alexa Fluor 350
From: Bill Telford (telfordw@box-t.nih.gov)
Date: Wed Jul 05 2000 - 14:50:05 EST
Hi Sheree...
We have had considerable experience with AMCA (7-aminomethylcoumarin)
over the years, both in home-brewed conjugations to antibodies and in
commercial preparations (avidin and anti-mouse IgG conjugations from
Molecular Probes, Jackson Immunoresearch, KPL, etc.). As I
understand it (if I'm wrong, someone at Molecular Probes corrct me!),
Alexa Fluor 350 dye is equivalent to AMCA-S, a sulfonated form of AMCA
that has higher aqueous solubility than conventional AMCA, giving better
conjugations - it also has a slightly shorter emission wavelength,
lowering interference with fluorescein. In any form, however, AMCA
is plagued by an emission wavelength coinciding with a region of strong
cellular autofluorescence, giving it a low signal-to-noise ratio.
Detection with AMCA by flow therefore tends to be very dim compared to
other synthetic fluorochromes such as fluorescein. We therefore
have only used it for extremely dense antigens (CD45/B220 on mouse B
cells and CD8 or CD90 on mouse T cells are typical examples) which would
give a FITC-detected signal of three or more log decades over background
- we would only expect one or two log decades above background for
AMCA. We used to use AMCA to do five-color analysis when we had an
Enterprise laser with 488 nm and 351 nm fixed wavelengths (along with
FITC/PE/PE-TXRed/PE-Cy5) - now that we have a 632 nm HeNe and a 407/415
nm krypton, we use either red-excited probes (APC, Cy5, APC-Cy7) or
violet excited probes (Cascade Blue or Cascade Yellow) almost exclusively
to get 5 or 6-color detection - they are all much brighter.
Now the good news! If AMCA-type fluorochromes DO work with your
detection system, your HeCad laser should excite it just as well as other
UV sources. We have used argon and krypton lasers ranging from 351
to 364 nm down to 20 mW power levels, and AMCA fluorescence over
background remains the same as at higher levels (150 mW). We also
recently tried Alexa Fluor 350 dye (MP avidin conjugate to detect CD90 on
mouse thymoma cells) on the new B-D LSR with its 8 mW HeCad source and it
worked just as well as a krypton laser emitting at 100 mW UV. The
data is at
http://www-dcs.nci.nih.gov/branches/medicine/flowcore/lsr.htm.
As you can see, though, the AMCA signal with any excitation source leaves
something to be desired - if you want to try it, some sort of
amplification might be in order (biotin-avidin or secondary antibody) and
would let you avoid the conjugation process.
Hope this helps.
Bill
(At 09:54 AM 7/5/00 +0930, Bailey, Sheree (FMC) wrote:
>
>
>
>We have a FACS Vantage SE with an Omnichrome HeCd laser with an
output power
>of 36mW.
>We would like to use Alexa Fluor 350 Dye and Protein Labeling Kit
with an
>Abs/Em
>of 346/442nm. Has anyone used this dye and/or this system and
do you
>recommend it.
>
>Sheree Bailey
>Flinders Medical Centre
>Dept of Immunology
>SA. Aust.
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