rhester at jaguar1.usouthal.edu
Tue Jun 29 14:19:31 EST 2004
[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.
Univ. of South Alabama
S Clark wrote:
>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
>Has anyone got any ideas as to what could be causing this problem, and how we
>can go about rectifying it??
>Flow Cytometry Technician
>University of Sheffield
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