From: colette charland (ccharlan@zoo.uvm.edu)
Date: Fri Apr 17 1998 - 17:17:12 EST
Greeting from the University of Vermont!
I have recently began to analyze specimens loaded with jc-1 and have
a few questions.
A little background info will be helpful. I have an Elite with a
488nm argon laser set at 15Mw. The "red vs green" signal although in a
upper right quadrant appears on a diaganol. STOP.... I have established
the appropriate compensation settings with a full range of FITC as well as
PE beads. ;-)
The cells are an adherent pulmonary mesothelial cell line, and we
are following a procedure as described by Dr. Cossarizza from the 3rd CD ROM
from Purdue.
The first question is why is the signal running on a diaganol. At
present the cells are being loaded with 150nM of JC-1. Is there not enough
of the probe or do I have to increase my laser output?
Secondly, although there have been changes in the fluorescent
signals relative to treatment with Il-1, TNF, H2O2, both the red and the
green decrease in intensity when read on a logarithmic scale.
This brings me to my third question which relates to ratios. Should
I establish a ratio parameter of linear fitc divided by linear pe, or should
the ratio fitc peak be divided by pe peak? Lastly, given that I've
successfully established the ratio parameter, should the investigator
monitor the change in the mean fluorescence of said ratio or just report the
peak channel value?
I have a number of queries that I would like to make regarding
probes in general, but I will save them for a later date.
Thanks in advance for all comments
Colette Charland
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