For inhibit intracellular calcium release you can use BPTA-AM. BAPTA is a Ca2+ chelator with 105-fold greater affinity for Ca2+ than for Mg2+. BAPTA-AM is a cell-permeable derivative which is widely used as an intracellular calcium sponge.

For trigger intracellular calcium release you can use for example, calcium ionophore A23187, a mobile ion-carrier that forms stable complexes with divalent cations. Useful for increasing intracellular Ca2+ levels.

Hi Sander,

ususally reagents like ionomycin or PMA are used to trigger Caclium flux. Both molecules oben intracellular Calclium storages and lead to strong Calcium signalling.

Greets, Mario

As Mario says, Ionomycin is very good to get an intracellular calcium increase! However, Ionomycin it is an ionophore and works by transporting calicum across the plasma membrane. I you don't mind how you get the calcium increase then this will be fine.

If it is calcium release that you are most interested in then Thapsigargin is a SERCA pump blocker http://www.sigmaaldrich.com/catalog/product/sigma/t9033?lang=en®ion=GB which is raise intracellular calcium levels, by preventing calcium uptake into ER and SR stores. I have looked at this in the context of T lymphocytes, addtionally you will get opening of calcium release activated calcium channels (CRAC [ORAI and STIM]).

Hope this helps, John

For inhibit intracellular calcium release you can use BPTA-AM. BAPTA is a Ca2+ chelator with 105-fold greater affinity for Ca2+ than for Mg2+. BAPTA-AM is a cell-permeable derivative which is widely used as an intracellular calcium sponge.

For trigger intracellular calcium release you can use for example, calcium ionophore A23187, a mobile ion-carrier that forms stable complexes with divalent cations. Useful for increasing intracellular Ca2+ levels.

Keep in mind that if you will use an ionophore, the cells must be in a buffer that contains calcium. If not, you will not observe an increasing in fluorescence.

Hi Sander,

An increase in intracellular Ca2+ can be seen by short-term (2 h) exposure to thapsigargin (10 nM) as well as the calcium ionophore A23187 (0.1 mM). You can also use pretreatments with diltiazem (0.01 mM), an inhibitor of calcium entry via voltage-gated calcium channels and chelation of extracellular Ca2+ by 0.005 mM) EDTA.

Hi Sander,

for Hl60 cells studying intracellular and intramitochondrial calcium level we have used (BAPTA-AM) (2–10 μM) or (EGTA-AM) (5–15 μM) as chelating agents and 0.1 μM thapsigargin and 1 μM ionomycin for triggering Ca2+ efflux.

Dear Sander,

There are many chemicals available in sigma which have been used to regulate calcium

some of them are 1. Cyclic adenosine diphosphate-ribose 2. Carboxyamidotriazole 3. 2-Aminoethyl diphenylborinate 4. Tyrphostin A9. and there ia another dye which can also be used for Ca2+ measurement, Quin-2, A fluorescent highly selective Ca++ indicator exhibiting fast uptake and release of Ca2+.

A note on mechanism. As mentioned, PMA and Ionomycin are often used in conjunction to get a synergistic calcium response, though for your purposes it may not be necessary to use both. Ionomycin, like Mariana said, is an ionophore (you can think of it like the natural CRAC that allows extracellular calcium into the cell). On the other hand, PMA essentially mimicks the action of DAG and activates PKC in the calcium cascade.

Thank you a lot for your very helpful advice!

Immune system cells usually mobilize calcium release through the IP3R pathway. You can block this calcium release by using xestopongin-C counteracting the IP3 pathway, or 2-APB inhibiting (but not specifically) the IP3-R. SERCA blockers such as CPA and thapsigargin will deplete the ER calcium stores, and will indirectly block calcium release through IP3-dependent stores. PMA by activating PKC will activate Receptor-operated channels such as TRPC and induce a calcium entry. You can also activate calcium entry through physiological agonists (chemokines) such as CXCL12 (SDF1-Alpha), also ATP, and may be thrombin. These agonists should activate both calcium release (first rising phase), followed by a sustained plateau due to calcium entry through PM calcium channels (SOC and ROC). good luck !

Which concentrations of BAPTA-AM are you usually using (and in which cell type)? What is the experimental setup - does it have to be i.e. preincubated overnight or something like this? Thank you!

thapsigargin (1-10 uM, 15 mins) or cyclopiazonic acid will empty intracellular stores and prevent release. 2-APB will inhibit IP3-mediated release. Could use fura-2/AM which is ratiometric and should give a reciprocal change in 340/380 fluorescence if effect is due to Ca2+.