Intracellular Calcium have become important indicators 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.
Calcium-signalling plays an important role in a number of physiological processes: For example nerves are triggered by Calcium-flux through voltage gated Calcium-channels and this subsequently leads to the release of neurotransmitters. In muscles the abrupt rise in cytosolic Calcium-levels culminates in contraction. In contrast, the rapid removal of Calcium into the sarcoplasmatic reticulum results in muscle relaxation. In the visual system Calcium controls the Guanylate cyclase after illumination. In the nucleus Calcium regulates transcription by activating the Calmodulin kinase which phosphorylates, and thereby activates the transcription factor CREB.

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 Centro´s, Mithras´s or TriStar´s reagent injectors.

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 or TriStar 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.

Calcium Assay Labels from Interchim

Fura-PE3 AM

• Calcium Assay with ratiometric dye
• Same wavelength as Fura-2
• Avoids problems associated with uneven dye distribution, cell or tissue thickness and photobleaching
• Reduced leakage

Fura-PE3 AM (500 µg)            FP-AM603B

Indo 1-PE3

• Calcium Assay with ratiometric dye
• Same wavelength as Indo1
• Avoids problems associated with uneven dye distribution, cell or tissue thickness and photobleaching
• Reduced leakage

Indo1-PE3 AM                       FP-AM602B

Fluo-4 AM

• Calcium sensitive assays
• Cell-based Calcium mobilization assay
• Special packaging for best storage

Fluo-4 AM (10 x 50 µg)           FP-729712
Fluo-4 AM (5 x 1 mg)              FP-729714