entire membrane fluor vs capped - equally bright by FACS?

Ray Hester rhester at jaguar1.usouthal.edu
Wed Sep 12 12:08:24 EDT 2007


An investigator has fluorescence microscopy data showing very bright, 
capped/polar fluorescence (present even at 4 C or with inclusion of 
azide in the FACS buffer) and yet the same cells (the investigator says 
they are 'polarized' cells) when examined by FACS are quite dim.  The 
FACS is a VantageSE and the fluorochrome is r-PE.  Can anyone tell us 
whether this should be the case or should a FACS detect capped 
fluorescence  just as efficiently as it does the same 'amount' of 
fluorescnce spread out over the entire cell membrane?

Certainly, 'bright' and 'dim' are relative terms, especially when you 
are comparing 'bright' fluorescence acquired with a fluorescence 
microscope to 'dim' fluorescence acquired with the FACS, so perhaps this 
should be treated as a theoretical question.   Also, fluorescence area 
has been collected to date on these samples; should they be acquiring, 
and comparing, fluorescence height as well?

Thanks for any help.

Ray Hester
Univ of South Alabama

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