Thanks for the feedback. You are probably right here. It would be nice if it could be made to work. We are working on permeabilizing ATP and know that at least some free luciferin can get into many types of cells. When we made the AM ester of luciferin it did not seem to improve things but if we could do both the luciferin AM and permeabilize ATP it might get this to work. I expect there should be enough Mg2+ in cells to get this started. Simon.Q.Rice@gsk.com wrote: > > I've tried observing cells using luciferase activity in the cytometer, > without success. > Luciferin requires ATP and molecular oxygen to drive the reaction, in > the presence of Mg2+. The difficulty I had (I think) was getting the > substrate (luciferin), and ATP, into the cells without damaging them. > Even using a caged luciferin, which is cell permeant and activated > intracellularly, didn't work. If anybody's got past that stage or > wants to discuss further off line, I'd be very keen to hear from them. > > Simon > > Simon QJ Rice > GlaxoSmithKline R&D > Harlow, UK > > > > "Richard Haugland" <richard.haugland@probes.com> > > 05-Dec-2002 03:02 > > > > > To: "Cytometry Mailing List" > > cc: > Subject: Re: luciferase essay using flow cytometer? > > > > > Interesting question. The chemiluminescence peak of luciferin is at > about 560 nm, which is a bit shorter wavelength than phycoerythrin > emssion. Of course, it does not need any excitation to get the emission. > But then, I don't know if the rate of emission will be fast enough to > get enough photons during the transit time that you will be able to > detect the signal. But then S/N should be great because all photons > should be "real." I don't know what technical difficulties there may be > doing sorting with the laser turned off but hope it works. > > > > > > Nan Jiang wrote: > > >Dear all: > > > >I am wondering if anybody ever sorted cells infected with luciferase > >plasmid. Or is there a particular wavelengh we can use to detect > >luciferase activity in cells and sort them based on that. > > > >Thanks in advance. > > > >Nan Jiang > > > >Department of Internal Medicine > >Cardiology Division > >UT Southwestern Medical center at Dallas > >6000 Harry Hines Blvd. > >Dallas, TX 75390-8573 > >(214) 648-1175 > >(214) 648-1181 > > > > > > > > > > >
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