Hi Sonja,
how many and what kind of lasers do you have?
Clontech claim an excitation maximum of 558 for RFP (DsRed), so if you've
got a 568nm Krypton line available, you should be able to separate it very
nicely from the GFP family on a two laser machine, but you'd need a more
selective orange filter.
If you've got two Argon lasers, you might try eCFP which excites with the
458 argon line (emitting around 480nm) alongside eYFP excited at 514 (or
488 - but watch out for noise from this in the eCFP detector).
If you've only got one 488nm Argon laser, I'd follow Derek's advice to use
a shorter wavelength for reading GFP, and be sure to use eGFP not eYFP
which might bleed more into the 585/42 filter. The document that Derek
quotes does show a histogram of FL2 using the 585/42 filter that you intend
to use <http://www.clontech.com/archive/OCT99UPD/images/RFPfig6.gif>, but
they "forgot" to label the x axis so it's hard to tell what the dynamic
range is.
I haven't tried this by flow either, and I'd be interested to know how you
get on as well,
Ray
At 9:03 pm +0000 9/1/00, Derek Davies wrote:
>Hi Sonja,
>
>According to the Clontech website
>(http://www.clontech.com/archive/OCT99UPD/RFP.html), the emission of
>RedFP is 583nm so it should be possible. If it is possible it may be an
>idea to change the GFP collection filter to something a little lower as
>the emission is 508nm (Should be possible if you are using a sorter or a
>FACSCalibur). I have no direct experience of this, but did do some work
>with the YFP which again was supposedly possible to use in combination
>with GFP but found it almost impossible due to the amount of spectral
>overlap; however, RFP may be a better bet. The quantum yield allegedly
>isn't as good as EGFP so I (and I am sure others) would be interested to
>hear how you get on.
>
>Good luck,
>Derek
>
>
>On Fri, 7 Jan 2000, Sonja Rotzoll wrote:
>> I want to detect by flow cytometry cells transfected with 2 vectors the
>> one containing the GFP protein and the other the new Red fluorescence
>> protein from Clontech (humanized Red1). Do you know whether it is possible
>> to measure them with this combination of filters:
>> PI 585/42 for reFP and
>> FITC 530/30 for GFP
>
>************************************************************************
>Derek Davies Voice: (44) 0171 269 3394
>FACS Laboratory, FAX: (44) 0171 269 3100
>Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk
>London, UK
>
>Web Page: http://www.icnet.uk/axp/facs/davies/index.html
>
>In tenebris lux
>*************************************************************************
Ray Hicks
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