RE: Luciferase detection

Unfortunately expression measurement only makes sense at the single cell
level.	I looked into a luciferase / GFP system to investigate
germination and a the luminometer measurement did not tell us anything
usefull. It could not distinguish high level expression of a few cells
from low level expression of many cells. Unfortunatelly both, the
luciferase and the GFP were independent plasmid systems.
Regards
Gerhard

________________________________

From: Robin Barclay [mailto:robin.barclay@ed.ac.uk]
Sent: Tue 19/12/2006 09:46
To: cyto-inbox
Subject: Re: Luciferase detection


Why don't you use a luminometer?
Robin Barclay

	----- Original Message ----- 
	From: Gagne Daniele <mailto:daniele.gagne@umontreal.ca>  
	To: Cytometry Mailing List
<mailto:cytometry@flowcyt.cyto.purdue.edu>  
	Sent: Friday, December 15, 2006 7:10 PM
	Subject: Luciferase detection


	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