Dear All,
For several months I have been trying unsuccessfully to detect the packard
GFP variants Topaz (under the control of a mammalian promoter) and Sapphire
(CMV promoter control vector) in several cell types using a fluorescence
microplate reader.
Has anyone out there had any success at all using a GFP reporter system on
a microplate reader to examine mammalian promoters, and can anyone offer me
any advice/help (other than give up and use luciferase/B-gal)?
The plate reader is the Cytofluor 4000 (Perkin Elmer). The cell types
include Hela, HepG2 and NIH-3T3s (all adherent cells).
Can anyone tell me the best culture plates to use for assaying GFP
expression in adherent cells (what materials do not autofluoresce across
the GFP wavelengths)?
Also can anyone tell me if standard any cell culture media components
fluoresce at around the GFP wavelengths?
Although I appreciate that other systems such as B-gal and luciferase offer
far greater sensitivity due to enzymatic amplification, the GFP approach
would be extremely useful for looking at the timescale of induction of the
promoter in question by a variety of stimuli. Since luciferase and B-gal
require cell lysis for assay a time-course experiment requires several
batches of cells, whereas GFP would allow the examination of the response
in the same cells.
Cheers in advance
Malcolm
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Dr. Malcolm S. Ogg
Centre for Cardiovascular Genetics
Department of Medicine
UCLMS
The Rayne Institute
5 University Street
London
WC1E 6JJ
Tel: 0171 209 6977
Fax: 0171 209 6212
Email: rmhamso at ucl.ac.uk
