Calcium ions are crucial for numerous cellular processes such as enzyme activity, muscle contraction, and neuronal signalling. Calcium assays employ either colorimetric or fluorescent techniques that measure cellular calcium concentrations.
The most commonly used system utilises recombinant jellyfish-derived calcium indicators, such as aequorin. Here are some uses of the calcium assay:
Cellular And Physiological Functions
The calcium ion is a ubiquitous second messenger in cell biology, modulating an enormous number of physiological pathways. It was first recognised as a crucial signalling molecule by Sydney Ringer, who demonstrated that saline solution made from London tap water supported contraction of frog hearts, while saline solution from distilled water did not.
A wide variety of cellular processes can stimulate the mobilisation of cytosolic calcium, including the binding of hormones to G-protein coupled receptors (GPCRs). This activation leads to generation of the second messenger inositol 1,4,5-triphosphate and releases calcium from intracellular stores.
Drug Effects
A calcium assay is a common tool to measure the activation state of ion channels and G-protein coupled receptors (GPCRs). Drugs that block or enhance these mechanisms often have dramatic effects on calcium concentrations.
Many synthetic calcium indicators are widely used due to their high stability, large dynamic range, and availability in a variety of spectral properties. Genetically encoded calcium indicators (GECIs) can be directly expressed in cells, avoiding the need for acute loading.
The sensitivity of a calcium indicator is dictated by its Kd value. Kd values for the most popular dyes, including Fura-2, Fluo-3 and Fluo-4, are around 10-100 nM at rest and can increase 100-fold at peak stimulation in certain cell types.
Molecular Mechanisms of Action
Calcium is a ubiquitous second messenger involved in everything from neurotransmitter release to muscle contraction to blood clotting and calcium homeostasis1. It interacts with a wide range of receptors, channels, transporters, and enzymes.
Changes in calcium concentration are an indirect marker of neural activity, but they can be correlated with action potentials and GPCR activation. Using a fluorescent calcium indicator such as Fura-2 or Fluo-3, researchers can monitor intracellular calcium by measuring shifts in fluorescence emission intensity caused by changes in the binding of the dye to cytoplasmic Ca2+.
Calcium Assay Kits
Several manufacturers offer kits for detecting calcium changes in cells. They come in a range of formulations and are designed to suit applications. They can be used to screen for GPCR and ion channel agonists that mobilize intracellular calcium stores, or to determine the inhibitory effects of compounds on calcium signals.
Synthetic calcium indicators are widely used due to their ease of use and wide dynamic range. Genetically-encoded calcium indicators (GECIs) have emerged to provide an alternative to synthetic indicators in certain applications. They offer greater sensitivity and a more natural signalling response but require stable expression to deliver reliable results.