determination of Ca intracelular

Dean Hewish deanh at tigger.mel.dbe.csiro.au
Thu Nov 16 21:35:53 EST 1995


I have tried the following protocol, originally given me by David
Hedley.
Fuo-3 is dissolved to 1mM in DMSO and added to cells in PBS to 5uM. The
cells are loaded with the dye for 120 minutes at 37 degrees then run
using a fluorescein setup.
Some cells have a p-glycoprotein membrane pump that will remove the dye
as fast as it loads. If loading doesn't work you have to inhibit the
pump with something like cyclosporin-A (3 micrograms/ml is the
concentration I have been given). Unfortunately, cyclosporin may mess
up the celular functions you are interested in, but there are other
p-glycoprotein inhibitors avaiable.
Fluo-3 only gives relative calcium concentrations since it is not
ratioable and can't be standardised easily

Dean R. Hewish
Cell biologist and Flow Cytometrist
CSIRO Division of Biomolecular Engineering
Parkville, Victoria, Australia



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