Cy-Chrome with dual-laser

Joe Trotter trotter at scripps.edu
Wed Nov 17 21:43:01 EST 1999


Dear Elena,

	I'm sure you'll get a lot of interesting responses. In short:
The tandem conjugate PE-Cy5 can be used with with both R-PE and APC
in a FACS Calibur *if* you set it up reasonably. Cy5 gets excited by
the red laser even though it is coupled with R-PE, so you compensate it
out of FL4 by sampling the FL3 signal excited by the primary 488nm
laser. The key is to have the HV "balanced" among the detectors such that
the CyChrome (+) population is brighter in the FL3 channel than in the
FL4 channel, otherwise it will not compensate. You can expect some
cross-talk with the FL2 channel as well, but the compensation is
straight forward if the detectors are set with similar
sensitivities as judged by where the (+) and (-) populations fall.
Since the PMTs are not all the same, you must use detector output to
guage where to set HV rather than the indicated numerical values in
the control panel. Since it is harder to get photons as you go out
towards the red, you can expect to use more HV there, especially
if you want to try to get backgrounds on scale.

The emission wavelength is essentially the same for Cy5 when excited by
the 633nm laser and when there is energy transfer from the R-PE. In the
Calibur, the 670nm LP in FL3 may see it a little better than the 661/16 BP
in FL4 because it passes more light. Since Cy5 emission is ~ 670nm, you
get a strong signal in both FL3 and FL4, however. We see differences among
batches of CyChrome, but have been able to use them all with FITC, R-PE,
and APC in a FACS Calibur. I'll leave it to others to elaborate more.
Mario's graphic at http://www.drmr.com/abcon/allspec.html may shed
some light on it for you after reading his notes on resonance
energy transfer dyes: http://www.drmr.com/abcon/noteRET.html.



	Good luck,

		Joe

On Wed, 17 Nov 1999, Elena Kalina wrote:

> 
> Ladies and gentlemen, 
> 
> it would be very kind of you to give me a detailed explanation to followed
> points:
> 1. It is possible to use simultaneously R-PE and Cy-Chrome with dual laser
> flow-cytometer (FACS Calibur BD)?
> 2. And APC with Cy-Chrome?
> 3. What is the problem by dubble-exitation of Cy-Chrome in detail (physic)?
> 4. How different are the emission-spectra of Cy-Chrome used with single and
> dual flow-cytometer?
> 
> Your expected answer would give us a grate help.
> 
> Sincerely Yours
> 
> Elena Kalina.
> University of Bonn, Germany
> 
> 

Joe Trotter 
Director, Flow Cytometry
Mailstop Imm-20
The Scripps Research Institute
10666 North Torrey Pines Rd.
La Jolla, California 92037





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