Dear Paul,
 
Calcium generally goes into the cell, so if big holes were punched in
the mebrane it would be goodbye cell. This does not happen so the
holes (if any) must be v.small. I still believe that the amount of
aldehyde is small, (not like bathing a cell in glutaraldehyde at
all)...
 
another $0.02 worth
 
Regards Mark
 
 
 
On Thu, 18 Jul 1996 10:12:26 -0700 Paul Goodwin wrote:
 
 
>
> OK, I'll confess stupidity here. It is my impression that aldehyde
> fixation crosslinks proteins (paraformaldehyde => single crosslink,
> gluteraldhyde => double crosslink). In our experience,
paraformaldehyde
> pokes holes in membranes, ostensibly by crosslinking surface
proteins, as
> is evidenced by the fact that we rarely need to detergent treat
cells to
> get anti-bodies in. Anti-bodies are big compared to Fluo-3. Why
don't you
> think that these holes are letting Fluo-3 and Ca++ out of the cell?
>
> Just my $0.02 worth.
>
>
______________________________________________________________________
__________
>
>
> Paul Goodwin
> Image Analysis Lab
> FHCRC, Seattle, WA
>
> On Thu, 18 Jul 1996, Natalie James wrote:
>
> > 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
> >
----------------------------------------------------------------------
-
> > Natalie James, PhD
> > CSIRO Division of Biomolecular Engineering
> > Riverside Corporate Park
> > PO Box 184
> > North Ryde NSW 2113, AUSTRALIA
> >
> > [log in to unmask]
> > (Ph) 61-2-886-4885 (Fax) 61-2-886-4818
> >