>We are attempting to measure pH within epithelial cells (HEC-1B) using
>C-SNAFL-1 Succinimidyl ester in the single excitation (514) dual emission
>(540/640) mode on our Zeiss CLSM.  We are having trouble generating a good
>standard curve for pH-clamped cells using nigericin/K+ to equilibrate
>the pH between buffer and cells.
 
Robert,
We found with single cultured cells (rat aotic smooth muscle and bovine
aortic endothelial cells) and snarf-1, we needed a concentration of about
20um nigericin to ensure "nice" standard curves. The concentration of
nigericin needed should depend on the mass of cell tissue in your culture.
Although you don't mention what concentration you are using, it might be
worth trying a higher concentration.
 
It is also very important to ensure that you focus into the middle of cells,
to exactly the same depth for each pH reading. You should use a fairly high
N.A. lens, in order to give a nice thin optical section. As you are measuring
different wavelengths in both channels (540/640), the optical sections
will not be perfectly aligned. If you have thick optical sections relative
to your cells, and don't focus to exactly the same point in the cells
you may find that at different measurements one channel may contain more
or less non-cellular information in your optical slice. This can leed to
great variability in standard curves.
 
See: Dubbin et al., (1993). Intracellular pH mapping with SNARF-1
and confocal microscopy. II: pH gradients within single cultured cells.
Micron, 24 (6), 581-586.
 
I hope this helps,
 
                                        __    /
   Stephen H. Cody,                   _/  \__/ \
   Department of Physiology,         /          \
   University of Melbourne,         /  Australia \
   Parkville, Victoria 3052,        \   ____     /
   Australia.                        \_/    \_*_/
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