Oops, of course it goes in. My mistake. But the holes that we are
observing must still be larger than an Ab so I can't see what would keep
the Fluo-3 in.
 
________________________________________________________________________________
 
 
Paul Goodwin
Image Analysis Lab
FHCRC, Seattle, WA
 
On Thu, 18 Jul 1996, Dr M Cannell wrote:
 
> 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
> > >
>