The importance of intracellular calcium measurement
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.
Measuring intracellular calcium using luminescence
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 (also known as 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 reagent injectors of the Centroluminometer and the Tristar microplate readers.
Measuring intracellular calcium using fluorescence
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 by 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 our multimode microplate readers is extremely fast (~150 ms), thus 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.
The fluorescent agent Fluo-4 can also be used to monitor intracellular calcium changes, and it has the advantage of using a single measurement, so no filter change is necessary. This is beneficial if changes in intracellular calcium levels are too fast for the instrument being used (for example, if it uses a monochromator or has slow filter change). However, it is not ratiometric, and hence it is more prone to varying assay conditions, leaking and bleaching.
Application Notes related to intracellular calcium
Calcium monitoring with the Mithras Calcium monitoring using Fura-2 with the Mithras LB 940 multimode plate reader
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Intracellular Ca2+ with Clonetics™ Primary Sensors and the Orion II Monitoring Intracellular Ca2+ Fluxes with Clonetics™ Primary Sensors using the Orion II Microplate Luminometer
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Screening of GPCR antagonists using Ca2+ measurements and the TriStar² S 3 commercially available compounds are screened for P2Y2 receptor inhibition using the calcium mobilisation assay with two different fluorescent dyes measured on a TriStar² LB 942 multimode microplate reader.
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Instruments recommended for intracellular calcium measurements
As mentioned above, injectors in measurement position are recommended for intracellular measurements using luminescence, and fast-switching filters for measurements using fluorescence. All microplate readers below fulfill those recommendations.
Tube luminometers equipped with injectors can also be used for intracellular calcium measurements using luminescence, and are also suitable if sample throughput is low. In tube luminometers, injectors are always in measurement position.