UV excitable dyes for surface phenotyping - ELF-97?

Adrian Smith A.Smith at centenary.usyd.edu.AU
Wed Mar 13 19:37:51 EST 2002


Here is some followup...

At 3:38 PM -0500 13/3/02, Gerstein, Rachel wrote:
>what wavelength light is the Spectrum laser capable of ?

The main lines are 647, 568, 530.9, 520.8, 514.5, 488, 457.9 & UV.

At 12:18 PM -0800 13/3/02, Andrew Beernink wrote:
>
>Is your second laser UV, or a HeNe?
>
>I get really strong lines at 457, 530, 568, and 647 nm.  I know the 647 line
>can excite a whole bunch of the AlexaFluor dyes (647, 660, 680, etc), at
>least two or three of which should be distinguishable.  If your Spectrum is
>UV rated, you might get usable violet (407, 413) light out of it, so you
>might try Cascade Yellow or Cascade Blue.  Check Mario Roederer's
>11-parameter immunophenotyping papers for a better idea.

The second laser is a HeNe and we are hoping to get two colours off
it - prime candidates at the moment are AlexaFluor 633 together with
AlexaFluor 660.

I don't think the Spectrum will give us usable violet so the Cascade
dyes are out.

  Mario's papers have been read several times here :)

At 11:58 AM -0800 13/3/02, Steven Sherwood wrote:
>On ELF, we used it to label endogenous alk phos in fixed cells. It worked
>extremely well once we understood that we had to cut back on the ELF
>concentration and time of reaction to prevent overly bright signals.

ELF-97 looks like a good candidate because it appears to be very
bright and emits past the autofluorescent maximum (it was tested here
when we were trialling an LSR last year). The fixation may be a
problem as we prefer to use live cells...but there are always
compromises.


At 3:31 PM -0800 13/3/02, Marty Bigos wrote:
>The original publications using cascade blue excited it with the UV
>from an argon laser. However, because of high autofluorescence, it
>was useful mainly as a second step with "bright" antibodies. We have
>used cascade blue conjugates with 407 nm krypton excitation; they
>are usable, but not great. Also, Molecular Probes makes Alexa Fluor
>350 conjugated as a second step reagent, which might be useful as
>well. We have no experience with it here.

Those were both on our list to test.

Thanks,

Adrian


--
______________________________________________
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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