On Wed, 27 Nov 1996 17:39:17 -0500, Andreas Rehm wrote:

>>I am a beginner in confocal laser scanning microscopy and I work in
>>calcium detection in the fungus 'Flammulina velutipes'. There are
>>hints in literature that calcium might be the secondary messenger in
>>the gravitropic reaction of the stipes of this fungus and I want to
>>find out if this is true by the means of confocal microscopy. Up to
>>now, I' ve employed the dyes 'Calcium green (AM)' and 'Fura red (AM)
>>-both from Molecular Probes. My problem is how to bring the dyes into
>>the cells! There are exoenzymes that seem to react with my dyes
>>before they are able to pass the membranes so that most of them
>>remain outside the cells........

On Wed, 27 Nov 1996 17:39:17 -0500, Milton Charlton wrote:

>We found in animal cells that some cells loaded some AM compounds better
>at reduced temperature.  Loading is a fight between extracellular
>esterases, intracellular esterases and anion pumps which dispose of the
>salt.  In addition to playing with temperature and different dyes you can
>also try blockers of acetylcholinesterase such as...........

We have also used "cold loading" to allow AM dyes to diffuse deep into
thick tissue, and also where there are extracellular esterases, with
success in animal and plant tissue. Incubating the tissue for longer at
temperatures as low as 0 Deg C at times. It is very important to always
give a post incubation period at 30 or 37 Deg C for at least 20 min. To
ensure that all the intracellular dye is cleaved before starting your
experiment. Otherwise your results may be erroneous if there is some
uncleaved dye in the cells.

See:

D.A. Williams, et al. (1993) Introducing and calibrating
fluorescent probes in cells and organelles. IN: Fluorescent and luminescent
probes for biological activity. A practical guide to technology for
quantitative real-time analysis. ED: W.T. Mason. Academic Press.

SH Cody, et al. (1993) Intracellular pH mapping with SNARF-1 and confocal
microscopy. I: A quantitative technique for living tissues and isolated
cells. Micron, V 24, 573-580.

Stephen H. Cody,                                        __    /
Biomedical Confocal Microscopy Research Centre        _/  \__/ \
Department of Pharmacology,                          /          \
University of Western Australia,                    /            \
Nedlands WA 6009,                                   \*  ____     /
Australia.                                           \_/    \_ _/
         email:  [log in to unmask]              __
         Phone:  61 09 346 4569   Fax: 61 09 346 3469         \/