Hello all,
I have a flow chamber to study calcium signalling in endothelial cells under
exposure to fluid flow (cell culture medium).  The indicator used is Fluo-3.
Dye loading of 1.5 um Fluo-3 for 20 min RT is used to give low levels of
intracellular dye, in order to minimise buffering of the calcium responses.
I have heard that there are potential fixation effects due to the aldehyde
degradation products of fluorescein-derived probes which are produced during
photobleaching, which occurs during scanning.  The original papers on the
development of Fluo-3 and its application in living cells (JBC 1989 (264)
8171-8 & 8179-84), don't discuss the possibility of this complication (& the
illumination was with a xenon lamp rather than a laser).
 
1.  Does anyone know whether aldehyde-related fixation is likely at the
concentrations of fluo-3 used for calcium measurements?
What intracellular concentrations of a fluorescein-derived compounds could
yield aldehydes that would give this problem?
Does anyone have references discussing this problem?
 
2.In addition this factor was suggested as a possible reason for loss of
contractility in experiments involving scanning of smooth muscle cells.  Any
comments?
 
I would very much appreciate any information on this question.
Thanks in advance.
 
Natalie James
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Natalie James, PhD
CSIRO Division of Biomolecular Engineering
Riverside Corporate Park
PO Box 184
North Ryde NSW 2113, AUSTRALIA
 
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(Ph) 61-2-886-4885 (Fax) 61-2-886-4818