Re: use of GFP and CFP reporters with a VantageSE

From: William Telford (TelfordW@mail.nih.gov)
Date: Wed Oct 23 2002 - 23:23:30 EST 




Hi Andrew...


CFP "likes" a violet or blue excitation source - krypton-ion
407 or 413 nm violet, or argon-ion 457 nm blue lines all provide
not-completely-optimal but decent excitation.  Unfortunately these
lines can't be had from an Enterprise II.  488 nm won't
work.


The cheapest solution for CFP on the Vantage is to obtain a violet laser
diode from Power Technologies or Coherent and set it up in the middle
(third) laser position, where your UV beam is now.  These relatively
low-power (10-20 mW) laser sources provide surprisingly good excitation
of CFP.  You will need to set up some sort of mounting system -
we've used a laser post mounting system from Newport.  You can set
up the whole system for a good bit less than $10,000 - much less than a
water-cooled gas laser. 


We have some sample data for violet laser diode excitation of CFP on our
website at
http://home.ncifcrf.gov/ccr/flowcore/index.htm
(direct access at http://home.ncifcrf.gov/ccr/flowcore/VDL.htm).  If you are interested
it trying this, we can send you more specific info.


Good luck,


Bill Telford

NCI-NIH


  At 12:11 PM 10/23/2002 +0100, Andrew Herman wrote:



Dear Flow-ers,

  I have a colleague who wishes to analyze and sort cells that bear GFP

and CFP (cyan fluor. protein) reporter constructs.

Has anyone had experience doing this type of analysis with a Vantage SE

and an Enterprise II laser (6 color config). I realize that using a 488

nm line is suboptimal for CFP, but is it feasible?


Any info would be very much appreciated.


Thanks,


----------------------

Andrew Herman

Department of Pathology & Microbiology

University of Bristol

University Walk

Bristol  BS8 1TD

Tel. +44 117 928 7511

Fax. +44 117 928 7896

A.Herman@bristol.ac.uk















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