Hi Gina
Do you mean just in your loading buffer or in the buffer you finally suspend your
cells in before running them? Chelating Calcium in your buffer you run your cells in
would mean any Calcium flux you see would be mobilization from intracellular stores. I
you want to see the flux accross the plasma membrane as well you will of course need
to have Calcium in your buffer. We use HEPES with 1mM calcium. You should probably
avoid having phenol red in your buffer.
Best of luck
Simon Monard
FACS Lab Manager
Trudeau Institute
Saranac Lake
NY12983
Ph 518 891 3080 X352
>>> <Gina_Graziani-Bowering@hc-sc.gc.ca> - 6/26/2001 5:09 PM >>>
Dear Flowers,
I am hoping that someone can clear up some confusion I have regarding the
presence/absence of calcium in buffers used to load cells in a calcium flux
assay. I have one protocol which says to use RPMI with 0.42 mM Ca2+. I have
seen other protocols in which chelators are added to the buffers to remove
calcium . Why the difference?
Thanks,
Gina
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