A calcium flux assay is a powerful tool to measure the agonist-stimulated and antagonist-inhibited signalling of G protein-coupled receptors (GPCRs), an important target class in drug discovery.
Fluorescent dyes can be loaded into cells to track changes in intracellular calcium. A radiometric calcium dye such as Indo-1, which requires UV excitation and emits at different wavelengths depending on its binding to calcium, is commonly used.
Detection Of Calcium Activation
During the calcium flux assay, cells are loaded with a calcium-sensitive fluorescent dye that is capable of crossing the cell membrane and binding to intracellular calcium. The change in fluorescence intensity is directly proportional to the amount of Ca2+ mobilized from the intracellular store and released into cytoplasm in response to agonist stimulation.
The sensitivity of the calcium indicators allows for detection of small changes in intracellular calcium levels and provides the information required to measure the activation state of ion channels and G-protein coupled receptors (GPCRs), an important target class in drug discovery. In addition, the ratiometric measurement offered by these systems reduces the effects of uneven dye loading, leakage of dye, or photobleaching.
Detection Of Calcium Inhibition
The cell’s ubiquitous second messenger, calcium, is involved in a wide range of physiological processes including nerve impulse transmission, muscle contraction and blood clotting. Changes in the concentration of calcium are transduced by a vast array of proteins and regulate various cellular responses1,2.
The fluorescent dye-based calcium flux assay is widely used for high throughput screening (HTS) of agonist-stimulated or antagonist-inhibited signalling through G protein-coupled receptors (GPCRs), which are a large target class relevant to drug discovery. A variety of ion indicators are available with intense fluorescence signals and a wide range of wavelength options, providing multiple possibilities for ratiometric measurements.
Detection Of Calcium Mobilisation
When an action potential arrives at the neuromuscular junction, it depolarizes the membrane, leading to a rapid influx of calcium through voltage-gated L-type calcium channels. This influx activates Ca2+-dependent contractile proteins and initiates muscle contraction.
A calcium flux assay is a cell-based second messenger assay that measures the mobilization of intracellular calcium after G protein-coupled receptor activation or inhibition. The assay is based on preloading cells with a calcium sensitive fluorescent dye that can cross the cell membrane. Once inside the cell, the dye binds to the released calcium from intracellular stores and its fluorescence intensity increases. The change in fluorescence intensity correlates to the amount of calcium released into cytoplasm in response to the ligand-activated receptor.
Calcium Transporters
The calcium ion is a ubiquitous second messenger and modulates an enormous number of physiological pathways. Hundreds of proteins have been developed to modulate these pathways, and many of them respond to changes in intracellular calcium levels, making monitoring the activity of these proteins an important tool for cell biology.
The Calcium Flux Assay uses a fluorescent ratiometric technique to monitor changes in the concentration of the ubiquitous cation. A non-washed cell sample is loaded with a series of esterified calcium dyes (probes) that can be cleaved to release their lipophilic blocking groups, allowing them to cross the membrane and become active upon binding to intracellular Ca2+.