Dear all, I wonder if any one can help me. I am currently measuring
intracellular calcium in EC using fura 2 with the improVision system. I have
been attempting to characterise the mechansims of the ca responses and have
came to the stage where I am looking at protein kinase C inhibition with
staurosporin (10-100nM). The stauro. is added after the cells have been loaded
with the dye (30 minutes) for 15 minutes and then I add my agonist of interest.
However when I come to look at the cells down the microscope they are
activated. Is this normal? Should inhibition of kinases cause activation of the
cells resulting in a rise in calcium? They also look rather purple looking, not
the green fluorescence one expects from fura 2. Also the staining occasionally
looks punctate. The dye however is not saturated as I can still produce a
response with Ionophore. Does this mean that although my cells are already
activated I can still look at agonist effects which I believe work through
Protein kinase C inhibtion with accuracy?