RE>calcium measurements                      5/2/96
 
Although I've been using indo-1 with confocal UV laser and published some of
the results, I would not recommend indo-1 as a first choice for gerenal use
for several reasons, some of which have been already pointed out here.
 
1. Because of difficulties in performing a reliable calibration, calculated
absolute Ca2+ values with indo-1 remain just an estimation in most cases.
Alternative quantitative measurement techniques with fluo-3 alone (e.g.
F/Frest) can be easier and more practical.
 
2. Quality of indo-1 ratio image is not as good as those obtained with fluo-3
alone or coloading method of fluo-3/fura-red.  This often makes a weak signal
like Ca2+ sparks in cardiac cells undetectable.
 
3. UV laser could significantly affect some cellular functions.  This is just
my impression through the experiments and remains to be confirmed, but it's
quite obviously seen in some cell types.  For instance, Ca2+ waves in cardiac
cells occurred less frequently during UV laser exposure than in visible
lights, and mast cells exhibited degranulation much less frequently in UV
laser than in fluo-3 settings.
 
4. UV laser and relavant confocal optics for it are costly.
 
5. The best indication for indo-1 which I come up with at the moment is some
particular cases where you want to see the Ca2+ differences between the
cytosol and the nucleus.  Fluo-3 seems to be quite significantly sequestered
into cytoplasmic organelles, which leads to underestimation of cytosolic Ca2+
compared to the nuclear Ca2+.  This would misleads to interpretation that
nuclear Ca2+ is higher than cytosolic Ca2+.  Indo-1 ratio image tells us that
the Ca2+ gradient in reality is just the opposite to it, eliminating the
sequestration artefact.
 
Anyway the best dye selection varies from case to case, depending upon the
purpose of the experiment.  However, if the flow chart for general guideline
is to be made, I would vote for fluo-3 alone as a first step to try because of
its simplicity and excellence in image quality.
 
Tets
 
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                           MINAMIKAWA Tetsuhiro
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