At 03:35 PM 7/18/96 EST, 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.
 
 
Dear Natalie,
 
The viability of mouse skeletal muscle is very good when loaded with
significantly more dye than you are using (i.e., 30 microM).  After loading
mouse skeletal muscle with 10 microM Fluo-3 AM and 20 microM Fura Red AM (30
minutes at room temperature), I have measured maximum tetanic force
production over a 2 hour period (20 degrees C).  There was a slight decrease
(6-8%) in maximum tetanic force output in the dye loaded muscles when
compared to unloaded control muscles.  However, I should also add that mouse
skeletal muscle has inherently low non-specific esterase activity which
reduces the total intracellular dye concentration to approximately 5 microM.
 
I hope this helps.
 
Chris.
 
 
 
 
 
 
 
 
 
 
Christopher P. Ingalls, Ph.D.
Post-Doctoral Research Associate
Muscle Biology Laboratory
Dept. of Health and Kinesiology
Texas A&M University
College Station, TX 77843-4243
U.S.A.
 
409.862.4808 (phone)
409.847.8987 (fax)
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