On Thu, 25 Jan 1996 03:49:47 -0800, M.J. Tobin wrote:
 
>We have recently started using the dye mixture of Fluo 3 and Fura Red to
>perform ratiometric measurements of calcium in living cells.  The literature
>suggested these would work well as they show equal distribution through the
>cells and little or no fading.  However I have been using viscous solutions of
>the non cell permeant equivalent of each dye to check the alignment of my
>instrument and have found that Fluo 3 bleaches very quickly.
>Has anybody else experienced this or has anyone a suggestion.
>
>Mark Tobin
 
This brings up an intersting point. The use of Fluo-3 and Fura-Red (FRed)
as a quatitative method (or semi-quatitative method), is based on these
assumptions:
 
That the dyes are evenly distributed in the cells.
That the dyes photobleach at the same rate.
 
While there has been suggestions in the literature that these assumptions
are acceptable, I cannot agree in all sitiuations that these assumption
will be true.
 
Using the same cell preparations (enzymatically isolated cardiac cells)
I've found dye loading varies from day to day and from cell to cell in a
single day. While in one cell on a particular day, you may get accumulation
of fluo-3 in the nucleus, the next day FRed may have accumulated. Or there
may be no accumulation. Working with single dye's, the rate of
photobleaching varies from day to day as well.
 
>From my experience the two assumptions are only rarely true, and then it
may only seem that the assumptions are fairly accurate. The dual dye
methods are not quantitative. You can use the dual dye method to confirm
that fluorescent changes seen are partially due to Ca2+ changes, but using
the ratio as a quantitative method is riddled with danger. Remember that
this is only a psuedo-quantitative method.
Stephen H. Cody,                                        __    /
Biomedical Confocal Microscopy Research Centre        _/  \__/ \
Department of Pharmacology,                          /          \
University of Western Australia,                    /            \
Nedlands WA 6009,                                   \*  ____     /
Australia.                                           \_/    \_ _/
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