I have been doing some blocker experiments to identify the mechanism of intracellular calcium elevation from US stimulation.
I used 10uM of BAPTA-AM, incubated for 20min, and wash with DPBS+.
and bathed with external solution(containing Ca2+) while stimulating.
With control (without any treatment) I could see calcium spikes from US stimulation however, in BAPTA-AM treated dishes I could not see any calcium activity.
Can I say that calcium elevation observed from control dishes was mainly from release of calcium storage (or at least Store-operated channel is responsible) other than calcium influx through one of machine-sensitive channels in membrane?
Hi Chi,
The fact that you can block US-induced calcium spikes with BAPTA-AM doesn't tell you about the source of calcium involved. To discriminate between a calcium entry or a calcium release from extracellular stores you could repeat you experiment in the absence of extracellular calcium (with maybe extracellular EGTA to really get 0 extracellular calcium) and see you you still observe your spikes. If yes, the effect is likely mediated by extracellular stores. Then you can try to block extracellular release, or deplete stores before US stimulation that is expected to abolish calcium spikes. But the very first thing for now is to discriminate between a calcium entry and a calcium release from stores - and you can't exclude a combined contribution of both, like a CICR effect of US stimulation.
Hope this helps. Good luck!
All you are doing is stopping any rise in calcium inside the cell, whether it is from intracellular stores or extracellular influx. BAPTA-AM cannot differentiation.
I agree with the depletion of extracellular calcium using 2 mM EGTA for 10 minutes prior to stimulation. This will tell you a component of the spike is from extracellular influx.
If you wanted to test whether the calcium spike is because of store operated release, then you should deplete the cells with 1 µM Thapsigargin for 10 minutes prior to stimulation. This will tell you if a component of the spike is from the intracellular stores.
It is important to acknowledge complete inhibition and partial inhibition, because often times there are multiple sources of a calcium flux.
If the calcium is mediated by intracellular stores, you should be able to observe calcium signals after thapsigargin treatment as has been suggested. And to underpin that intracellular stores are involved rather than entry of Calcium from outside, you could try loading simultaniously with BAPTA-AM in "Calcium free" buffer. Thereby you exclude that Calcium enters the cell from the outside and if then with BAPTA-AM the signal mediated by thapsigargin disappears you have a strong evidence for intracellular stores mediated Calcium release
I agree with most of the insightful comments posted, but would caution leaving cells in media with no extracellular calcium. Some cells hang on to their stored calcium for a long time when they are incubated with a 0 calcium-solution, but others (notably excitable cells) do not. Sitting cells in an extracellular medium with no calcium for a long time will lead to depletion of intracellular calcium stores, trigger a stress response, and confound the interpretation of your experiments.
Moreover, some cells don't like 0 calcium solutions at all. In which case, another way to go is to use a blocker of calcium influx such as lanthanum, gadolinium or another specific compound if you are aware of the putative calcium entry pathway.
Thapsigargin is a useful tool (we prefer cyclopiazonic acid as it's much easier to use being lower affinity and reversible), but if you think that there is a release of calcium from intracellular stores then ryanodine, 2-APB, expression of the InsP3 5'-phosphatase and other channel-blocking compounds can be used.
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