From: maciej simm (simm@cd4cd8.com)
Date: Mon Dec 09 2002 - 16:06:24 EST
I use a Cylex luminometer to detect ATP of leukocyte lysates. Combined with magnetic bead isolation of cells we get pretty good measurements of specific populations. It is not as fast and convenient as flow but it works pretty reliably. I'll be happy to answer questions about this gadget to anyone interested. I am not an employee of cylex nor do I receive any payment from them.
Maciej
----- Original Message -----
From: David Galbraith
To: Cytometry Mailing List
Sent: Friday, December 06, 2002 3:47 PM
Subject: Re: luciferase essay using flow cytometer?
This question seems to come around at regular intervals! As Dick notes, the problem is that even transgenic organisms expressing large amounts of luciferase do not produce sufficient photons per second for a significant number to be collected by the flow cytometer during the (very short) period of time that the cell is in the focal point of the collection lens. This is compounded by the fact that the light is emitted in all directions, and the efficiency of collection of light by the flow cytometer is nowhere near 100%. If you were to slow down the cells, this would work (as of course does conventional imaging of luciferase in transgenic organisms under static conditions!). however, my guess is the cells would have to be very nearly stationary....
Best wishes
David
At 08:34 AM 12/6/2002 +0000, 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
>
>
>
David W. Galbraith
Professor of Plant Sciences
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://latin.arizona.edu/galbraith
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