UV excitable dyes for surface phenotyping - ELF-97?

William Telford TelfordW at mail.nih.gov
Mon Mar 25 01:52:47 EST 2002


Hi Adrian...

Sorry for taking so long to respond to this, my computer/e-mail has been
very shaky lately.

The protocols we describe for using ELF-97 for immunophenotyping in our
Cytometry papers are now a little dated - we are in the process of writing
up a communication with updated info. For immunolabeling, we no longer use
the kit buffers but just dilute the substrate in 2% FBS in PBS and add it
to the AP-labeled cells for 15-30 minutes (4C is OK). The "problem" with
the kit buffers is that they attempt to minimize the particle size of the
ELF precipitate, for better visual localization of antigen in cells and
tissue sections by microscopy. To generate this type of precipitate, MP
uses buffers with salt concentrations well above isotonic - while they
accomplish their intended aim, these conditions are hard on live cells and
can cause clumping in some cell types. The particulate size does not matter
for flow, and we have found that using simple PBS with protein gives almost
the same level of fluorescence as with the kit reagents. Adding 2% FBS
reduces cell clumping for us. After talking to Molecular Probes, we have
experimented with adjusting the Zn2+ and Mg2+ levels in the labeling buffer
to increase substrate brightness - it helps a bit, but there is probably
enough divalent cations in serum normally used for immunophenotyping to
give good substrate reactivity.

Live cells labeled with ELF-97 fix OK with formaldehyde - you can also fix
the cells following AP labeling then followed by ELF-97 treatment, with a
small reduction in fluorescence. ELF labeling is spectrally compatible with
FITC, PE, PE-Cy5 and so can be used in multicolor flow quite easily. You
can buy the ELF-97 substrate alone from Molecular Probes. We have labeled
cells using biotin-anti-something, then AP-streptavidin and ELF. We also
have some AP-conjugated anti-heat shock protein antibodies that work well
for flow - we get better sensitivity than with indirect labeling with FITC.
We are also trying to make some AP-direct conjugates - not as easy as we
were hoping, unfortunately!   An important warning- AP has strong
non-specific binding to some cell types.  Incorporating 1% dried milk into
your labeling buffer during the AP labeling will help prevent this - just
like a Western blot!

We use a standard 530/30 FITC filter for detecting ELF. Any UV laser works
fine - we have compared a krypton-ion 351 nm (100 mW) on our Vantage with a
HeCad 325 nm (8 mW) on an LSR and they seem to work equally well. Also, if
you have a violet krypton laser line (407 nm), you can do Cascade Blue and
ELF-97 together (somewhat reduced fluorescence for ELF, though, since you
are only catching the tail of its excitation spectra). Even our little 408
nm violet diode laser excites ELF-97 reasonably well, so your mixed-gas
laser should work just fine at either UV or ifv necessary, violet
excitation.  With the UV line, AMCA, Alexa Fluor 350 and DAPI/Hoechst dye
all are compatible with ELF. To do ELF and a blue probe like DAPI or Alexa
Fluor 350 or AMCA together, we use a long pass dichroic (460 LP or 480 LP)
to split the signals.

Hope this helps.  I'll be happy to send you some preliminary data is you
are interested.

Take care,

Bill Telford
NCI-NIH



At 12:31 PM 3/13/2002 +1100, Adrian Smith wrote:

>Hi all,
>         We are in the process of getting some extra colours working
>on our new FACSVantage SE and are trying to find a dye we can excite
>with the third laser (a mixed gas argon and krypton (Coherent Innova
>70 Spectrum UV)).
>
>I have read the Molecular Probes literatures and the publication by
>Telford et al (Cytometry 32:117-125 (2001)) about the use of the
>ELF-97 enzyme substrate system and was wondering if anyone has any
>"real-world" experience using it with surface antigens? I am
>particularly concerned about the 30min incubation at RT that is
>required. What does this do to live cells? (the MP literature
>suggests using fixed cells but the Cytometry paper seems to use live
>cells). What about the labelling of other surface markers?
>
>Any other suggestions for alternative dyes that we can use with this laser?
>
>Thanks,
>
>Adrian Smith
>
>--
>______________________________________________
>Adrian Smith (Research Officer)        T CELL BIOLOGY GROUP
>Centenary Institute of Cancer Medicine & Cell Biology
>Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA.
>Ph: (02) 9565-6198 Fax: (02)-9565-6103





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