Hi all,
 
With all these cents flying around I thought I'd spend a penny:)
 
The way I read the original letter, she isn't using aldehydes to fix her
cells in any way,  she's worried that photolysis products of FLUO-3 may
contain aldehydes, and that those aldehydes may be at a concentration that
will interefere with the cells' physiology.
 
It's quite a tough one to answer, since although it's possible to bathe
cells in varying external concentrations of aldehyde and see what happens,
the conentration needed to fix and permeablise the membrane may be much
higher than that required to disrupt a metabolic pathway.  Intracellular
aldehyde may well be a problem even at low concentrations, but it's hard to
imagine (for me at least) how you might go about introducing known amounts
into a viable cell with intact membranes to study their effect.
 
It's also hard to tell whether the aldehydes would have as large an effect
as the free radicals they might derive from.
 
 
 
 
Ray
 
                              Ray Hicks
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At 4:49 pm 18/7/96, Paul Goodwin wrote:
>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
>> > >
>>