Hi David,

are the 37 degrees the problem? If not already done, try loading cells
with the AM ester at room temperature. There are cells that load well at
room temperature, maybe you have to double the dye concentration and/or
incubation time. If you are using the cells for confocal calcium imaging
(excitation at 488 nm), I would recommend Fluo-4. You need only half the
amount of dye in the cells to get the same fluorescence intensity as
Fluo-3 at 488 nm. In our lab in Kaiserslautern, Germany, Fluo-4-loading
works fine at room temparature in astrocytes in culture.

Cheers
Chris


David Driver wrote:
>
> Greetings,
>
> I was wondering if anyone out there had experience in electroporating
> calcium indicators into mammalian cells.  We would like to load Fluo3 into
> lymphocytes, avoiding the 37 degree loading step required to load cells
> with the AM-ester form.
>
> Alternatively, if anyone has any suggestions regarding other kinder and
> gentler methods to move Fluo3 into cells, I'd appreciate the suggestions.
>
> Thanks,
> David
>
> ______________________________
> David Driver
> Dept. of Immunology
> Duke University Medical Center
> Box 3010 DUMC
> Jones Bldg.
> Durham, NC  27710
> 919.613.7822
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> _______________________________

--
Christian Lohr, Ph.D.
ARL Division of Neurobiology
University of Arizona
PO Box 210077
Tucson, AZ 85721-0077

Phone: (520) 621-6671
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