Intracellular Calcium have become an important indicator for the activation state of ion channels and G-protein coupled receptors as well as for the phases of apoptosis and cell injury. Though the respective kinetics and the absolute amounts of the Calcium levels are different for each of these physiological processes there are common ways for monitoring them.
Bivalent Calcium is an intracellular messenger in many eukaryotic signal transduction pathways. Most Calcium-signalling systems have one thing in common: they generate brief pulses of Calcium, thereby regulating cellular functions.
Intracellular levels of Calcium are usually kept low, as Calcium often forms insoluble complexes with phosphorylated and carboxylated compounds. Typically cytosolic Calcium-concentrations are 100 nM. In response to stimuli Calcium is either released from external medium or internal stores to raise the Calcium-concentration.
Luminescenence Calcium Monitoring
The monitoring of the changes in intracellular Calcium levels by the means of aequorin in a flash-type luminescent reaction is highly efficient. Assays for both, the activation of G-protein coupled receptors (GPCRs or 7TM receptors) inducing inositol phospholipid degradation and the activity of voltage gated Calcium-ion channels - the latter causing a rather slow influx – can easily be performed with the use of the regent injectors of the Centro, Mithras or TriStar microplate readers.
Fluorescence Calcium Monitoring
Also the concentration of free Calcium in intact cells can be monitored by using polycyclic chelators such as Fura-2 or Indo-1. Fura-2 and Indo-1 provide a ratiometric readout thereby reducing effects caused by leaking or bleached dyes or varying assay conditions.
The fluorescence properties of both dyes are markedly changed when Calcium is bound and thus the direct detection of Calcium-fluxes in response to specific signal transduction pathways is possible.
The emission maximum from Indo-1 shifts from ~475 nm in Calcium-free medium to ~400 nm when the dye is saturated with Calcium. With Fura-2 the absorption maximum of Fura-2 shifts from 380 nm to 340 nm upon binding Calcium, while the emission remains constant at 510 nm. This results in an opposite change of fluorescence intensity: an increase at 340 nm and a decrease at 380 nm.
The filter change in Mithras is extremely fast (~150 ms) enabling a high resolution of detection when fast changes in Calcium concentration are to be monitored. Using at least one injector located in reading position fast reaction kinetics can be monitored.