On Thu, 18 Jul 1996 15:35:01 EST Natalie James wrote:
 
 
Dear Natalie,
 
I have not heard of any fixation effects of using the AM derivatives.
We have never anything in cell responses that could not be
explained by the calcium buffering. It is likely that the relatively
small amounts of aldehyde produced by de-esterification are lost from
the cell or destroyed by cell metabolism. In the worst case, there is
so much cell protein present that the limited quatities of aldehydes
would not be able to 'fix' the cell...
 
In summary, I wouldn't worry too much about fixation unless you
see an effect that is not explainable by the calcium buffering.
 
In addition , I have never noticed a large loss of cell
viability on exposure to the AM dyes.
 
I hope this view is endorsed by the group...
 
Regards Mark Cannell
 
> 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
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