Hi everyone: This question does come around regularly! Has anyone produced a bifunctional GFP-luciferase fusion? Then we could do a head-to-head comparison of sensitivity using all the available different assay platforms. In short, the time-of-flight of a cell through a flow cytometer is too rapid to allow meaningful collection of luciferase-mediated photons, despite its exquisite sensitivity over long times of collection of light in imaging modalities. Going to an antibody-based assay would mean that you lose the advantage of a flow assay in vivo, and probably that you would lose sensitivity (at least relative to GFP or its colleagues). Howard can probably chime in here as well regarding slow-flow (Happy BD by the way, Howard!). David At 12:10 PM 12/15/2006, Gagne Daniele wrote: >Hi everyone, > >I’ve been asked to post this question about luciferase detection: > >« Luciferase reporter gene expression can be >detected and quantified with very high >sensitivity in bioluminescence tests from whole >wells containing cells or organs. However, it is >not useful when evaluating events at the single >cell level, due principally to the low emission >intensity of the substrate luciferin. I read the >thread of Nan Jiang dec 2002 but was wondering >if things had evolved since then. In other >words, I would like to know if someone has found >a way of differentiating luciferase positive and >luciferase negative cells by cytometry: either >in microscopy or flow. It could be by the direct >detection of activated luciferin substrate, but >also by using antibodies against luciferase. > From my own searches, there are few antibodies >(so if someone already knows of a nice working >one for FACS) and we found one protocol >suggesting a 10 min acquisition under the >microscope to detect the activated substrate. Any help would be appreciated, > >Thank you! > > >Danièle >Danièle Gagné >Conseillère Technique Cytométrie >IRIC, Université de Montréal >Bureau 1404, Pavillon Marcelle-Coutu >Tél.: (514) 343-6111 x1-8094 >Fax: (514) 343-7780 > David W. Galbraith Professor of Plant Sciences & Professor, Bio5 Institute University of Arizona Office: 822D Marley Building Mailing address: Department of Plant Sciences University of Arizona 303 Forbes Building P.O. Box 210036 Tucson Arizona 85721-0036 USA. Tel: (520) 621-9153 Fax: (520) 621-7186 Email: galbraith@arizona.edu http://cals.arizona.edu/galbraithReceived on Tue Dec 19 11:58:00 2006
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