Using a fluorescent calcium indicator, this calcium flux assay measures mobilization of intracellular calcium. It is easy to perform in high-throughput applications and requires only one dye addition. It also does not require washing and can be used on dual-dispensing microplate readers. It also requires the addition of probenecid, a standard inhibitor of nonspecific anion transport, to improve retention of the dye in cells.
Calcium Flux Assay
Calcium plays an essential role in cellular physiology, including signalling pathways, neurotransmitter release, muscle contraction, enzyme activity, fertilization, and bone formation. The cytoplasmic concentration of calcium is tightly regulated, and imbalances can cause a variety of pathological disorders.
G protein-coupled receptors (GPCRs) are the major class of membrane proteins involved in regulating calcium homeostasis. Inhibitors of GPCRs may be potent for the treatment of several diseases. Among these are inflammatory, neurological, and endocrine diseases.
Fluo-8
Fluo-8 is a fluorescent calcium indicator that can be loaded into GPCR expressing cells. This indicator is lipophilic and can cross the cell membrane, where it binds to intracellular-released Ca2+, enhancing its fluorescence. The indicator is paired with a quencher dye, which masks excess calcium present in the medium. This allows the fluorescence signal from the calcium binding to be measured.
Fluo-4
GPCR-mediated calcium flux assays are the preferred method for determining agonist-stimulated and antagonist-inhibited signalling through G protein coupled receptors (GPCR) in cell-based high-throughput screening (HTS). These assays utilize an intracellular fluorescent dye that is activated by GPCR activation to monitor calcium mobilization.
In these assays, cells expressing GPCRs that signal through calcium are pre-loaded with the fluorescent indicator Fluo-4 AM. The lipophilic blocking groups of the AM ester are cleaved by non-specific cell esterase, leaving free fluorescent Dye that is ectopically bound to internal calcium upon stimulation.
Cal-520
Cal-520 AM is a new fluorescent calcium indicator with improved brightness, Fmax/F0 ratio and intracellular retention compared to Fluo-4 AM. It has also been shown to reliably detect spontaneous calcium transients in mouse barrel cortex neurons.
Cells are bolus loaded with ci-IP3-PM (a caged cell permeable analog of IP3) and Cal-520 AM and imaged by TIRF microscopy. The indicator is photo-uncaged by near UV light to induce Ca2+ release in cells.
Calbryte 520
Calbryte 520, 590 and 630 are a series of fluorescent calcium indicators designed for use in high throughput screening of G protein coupled receptors (GPCRs) and other cellular assays. These novel dyes are probenecid free and provide a very robust fluorescence signal when bound to intracellular calcium.
Unlike other calcium indicators, these dyes are soluble in DMSO and can be loaded directly into live cells. The long Ex/Em wavelengths of these dyes allow them to be easily excited at 488 nm and detectable using standard fluorescence instruments.
