Dear Confocalist

I would like to visualize real-time transcriptional activity using the
promomter fused upstream of luciferase cDNA (plasmid pGL3 basic;
Promega) in living cells using photon counting imaging after transiently
transfected with plasmids. We have BioRad MRC 1024 (both inverted and
upright, Kr/Ar and Ti:Sapphire laser). The reference I have does not
explain detail and some of key references are missing from the library,
unfortunately. Has anyone used this technique? Could anyone give me some
suggestions, considerations, protocols? Especially the methods regarding
to how to collect pGL firefly luciferase signal (transcriptional
activity) and Renilla reniformis luciferase signal (as control)
separately in living condition, how to expose to luciferin (incubate
with DMSO?, how long stable?), and what filter to use to collect image
etc. (Since I am collecting luminescence, I guess I do not have to
excite, right?)
Thank you for your help!!

Shuko

--
Shuko Harada, M.D.
Assistant Professor, Research
Dept. of Pathology and Lab Medicine
University of Pennsylvania School of Medicine
D101 Richards 37th Hamilton Walk
Philadelphia, PA 19104
Phone: 215-898-6738
Fax: 215-573-2350