GFP transfection

Ray Hester rhester at jaguar1.usouthal.edu
Tue Jun 29 14:19:31 EST 2004


Sue,

[Afer re-reading your question, I realize this isn't going to apply to 
your situation (since they see the fluorescence - by FACS? - when 
anti-GFP antibody is added) but maybe it will be applicable to someone 
out there.  Hope so.]

I would believe the FACS before I believed the scope.  Many fluorescence 
microscopes have filters selective for excitation light but not emission 
fluorescence.  As a result, everything above, for example, 500 nm light 
(when using the FITC/GFP filter cube) can be seen (these microscopes 
depend on the correct excitation light wavelength to produce the 'right' 
emitted fluorescence. Investigators will sometimes look at a sample 
under the fluorescence scope and see, e.g., 'green fluorescence' but 
when they analyze the same sample by FACS (or, in our lab, the Leica TCS 
SP2 confocal system), they fail to see this fluorescence.  And it's 
because the FACS probably has a 530/30 band pass filter in front of the 
green PMT (our confocal has somewhat similar 'filters') and what they 
are seeingin the scoppe is of a different emission wavelength - it's 
something, all right, but just not FITC (or GFP).  Often this non-green 
fluorescence (at least when i see it in the fluorescence scope) is more 
of a yellow/orange tint.  I tell them, hopefully correctly, if	it's not 
lime green in the scope, it's not FITC/GFP.

Of course you can buy cubes for fluorescence microscopes that have 
emission band pass filters, but I believe they're more expensive than 
the ones I mentioned above.

Hope this helps.

Ray Hester
Univ. of South Alabama
Mobile, AL




S Clark wrote:

>Hello there
>
>One of my customers is having difficulty with a GFP assay. The GFP is virus
>conjugated and following transfection, the cells are all green when looked at
>through a microscope, however when looked at on the FACSort they flourescence
>is less than that of the untransfected cells.
>
>They have added and anti-GFP antibody which stains positive, so it is obviously
>there.
>
>Has anyone got any ideas as to what could be causing this problem, and how we
>can go about rectifying it??
>
>Thankyou 
>
>Sue Clark
>Flow Cytometry Technician
>ICS, DGM
>University of Sheffield
>  
>




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