Hello everyone,
Recently I requested some clarification on why various Ca2+ flux assay
protocols differed with respect to the presence or absence of calcium in the
loading buffer used, ie. In some protocols, calcium was a component of the
buffer, while in others, calcium was not.
Below is a summary of the responses I received. Many thanks to all of the
respondents!
Doug Smoot wrote:
As I understand it, you use Calcium chelators to measure the use of
intracellular calcium stores. Otherwise, you are measureing the component
of the extracellular calcium the cell uptakes. For cell activation, there
are 2 components to the calcium activation, the intracellular stores being
utilized which often happens first, then the uptake of calcium from the
external media. My former boss, Dr. Carl June, has written several papers
on this, along with Dr. Peter Robinovitch, not to mention the many others
who have used calcium analysis. I hope that this helps.
Andrew Beernink wrote:
I know in our work, some ligands open Ca2+ channels, while some release
intracellular Ca2+ stores. Ca2+ in the medium would affect the former, but
not the latter, I would assume.
Sonja Rotzoll wrote:
If you use buffer with Ca you can see the intracelluar and extracelluar
flux of Ca. If you use buffer with chelators you can see only the
intracelluar flux of Ca. But you need ca in your buffer to see any signal
(so you use it with chelators)!
Nigel Miller wrote:
When you do Ca++ flux measurements it is sometimes necessary
to do a 'max' and a 'min' . My guess is that this is what you have seen.
With a max we give excess Ca and ionomycin and with a min we saturate with
EDTA or EGTA(specific for Ca at all pH's) and ionomycin.These expts are
always performed at the end of the day. When you do the flux expts( we use
Indo 1) you call on two Ca pools -one internal and one extracellular.You
never add a chelator when doing your runs.
Simon Monard wrote:
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.
Thanks again,
Gina
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