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Some people in my lab have started dabbling with GFP recently, they've used
Clontech's humanised "red shifted" (ie 488 excitable) gfp construct, and
the fluorescence is remarkably bright using the standard FITC filter on a
Vantage. One person transiently transfected it into COS, another put it
into human fibroblasts, in both cases we got off scale fluorescence in some
of the cells using 100mW of laser light (488nm). The negative cells stayed
put in the first decade of the log FL1 axis, the GFP showed up as a wide
CV'd spread starting just past the negative distribution, and continuing up
to the maximum channel where a reasonable number piled up.
You might well get better results using one of these new constructs than
you would by exciting wild type GFP at 413 (although using 413 should
improve what you're getting at the moment)
Ray
At 7:33 am 13/11/96, Pizzo,Eugene wrote:
>All Colleagues,
>
>A researcher here at UCONN is considering using the 413nm violet line
>of a Krypton laser to excite mutant GFP. This researcher has been
>somewhat dissatisfied with our results using 488nm excitation on the
>FACStar Plus (by the way we've been using a 500 EFLP in Fl1 to
>capture the emission). Can anyone enlighten me as to the worthiness
>of this approach? Is there a new GFP out there or is 413nm excitation
>really significantly better?
>
>Gene Pizzo/UCONN Health
Ray Hicks
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
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