Calcium influx assays
8 likes3,659 views
This document describes various calcium influx assays that can be used to measure calcium flux in cells. It discusses fluorescence-based assays that use calcium-sensitive fluorescent dyes like Fluo-8 and Rhod-4 to detect increases in intracellular calcium levels. Luminescence-based assays using aequorin are also described. The assays can be used to study G-protein coupled receptors and calcium channels by detecting changes in calcium mobilization when cells are stimulated with agonists. Ratiometric analysis methods that detect both increasing and decreasing fluorescence signals provide quantitative measurements of intracellular calcium levels.
Calcium influx assays
- 1. Calcium Influx Assays Department of Pharmacology
- 2. Calcium Influx Assays • Calcium mobilization assays is a cell based second messenger assay to measure the calcium flux associated with Gq-protein coupled receptor or inhibition or activation. • The method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. • In some cells anion transporter are particularly active. • The addition of probencid, an inhibitor of anion transporter, is required for retention of this dye in the cell. • The dye binds the calcium released from intracellular store and its fluorescence intensity increases. • The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of receptor
- 4. Luminometric Assay • It is much more sensitive than the fluorescence-based calcium assays. • It provides an optimized assay method for monitoring the G- protein-coupled receptors and calcium channels • Useful for monitoring the intracellular calcium mobilization in a specified compartment given that recombinant apoaequorin proteins. • More sensitive than the fluorescent calcium assays
- 5. Principle • Luminometric assay are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). • It uses a highly calcium- sensitive and membrane- permeable coelenterazine-calcium indicator for the cells that are transfected with apoaequorin gene. • Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea victoria that has been used extensively as a calcium indicator in cells • The aequorin complex emits blue light when bound to calcium ions. • The luminescence intensity is directly proportional to the Ca2+ concentration
- 6. Fluorometric Assay • Fluorometric assay provides a simple method for detecting calcium in physiological solutions by using a red fluorescence probe. • The fluorescence signal can be easily read by a fluorescence microplate reader at Ex/Em = 540/590 nm Fluo-8 Calcium Assay • It is a fluorescence-based assay for detecting intracellular calcium mobilization. • It provides an optimized assay method for monitoring the G- protein-coupled receptors and calcium channels
- 7. Principle • Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fluo-8 which can cross the cell membrane. • Once inside the cell, the lipophilic blocking groups of Fluo-8 are cleaved by an esterase, resulting in a negatively charged fluorescent dye that stays inside the cell. • Its fluorescence is greatly enhanced upon binding to calcium. • When cells are stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increases the fluorescence of Fluo-8.
- 8. Rhod-4 Calcium Assay • It is a fluorescence-based assay for detecting intracellular calcium mobilization. • Compared to Fluo-8, Rhod-4 is more photostable, making its fluorescence imaging more robust. Principle • Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Rhod-4 which can cross the cell membrane • Once inside the cell, the lipophilic blocking groups of Rhod-4 are cleaved by an esterase, resulting in a negatively charged fluorescent dye that stays inside the cell.
- 9. Cal-520, AM Assay • It provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization. • Cal-520 AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM)
- 10. Principle • Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520 AM which can cross cell membrane. • Once inside the cell, the lipophilic blocking groups of Cal 520 AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. • Its fluorescence is greatly enhanced upon binding to calcium. • When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520. • The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520 AM an ideal indicator for the measurement of cellular calcium.
- 11. Ratiometric Analysis • Ratiometric analysis of a calcium dye involves detecting an increasing fluorescence signal as calcium is released into the cell cytoplasm. • In combination with a decreasing signal as fluorescence is quenched by cytoplasmic calcium
- 12. Optical nanosensors and ratiometric imaging • Optical nanoparticle (NP)-based sensors used as tools for detection of ions and biomolecules. • It includes use of different sensing components. Greater target specificity and the ability to tune the dynamic range Encapsulation of multiple dyes to generate a ratiometric signal with varying spectra. The nanosensor incorporates highly calcium-selective ionophores and two fluorescence indicators that act as signal transducers to facilitate quantitative ratiometric imaging
- 13. • The nanosensors are fabricated by co-immobilizing hydrophobic optode components comprised of calcium ionophore, fluorescent chromo-ionophore,ion exchanger and reference fluorophore within a biocompatible plasticizer nano-emulsion matrix. • Stabilized by a biocompatible polyethylene glycol (PEG)-lipid as surfactant. • The hydrodynamic diameter of nanosensors measured 67 nm (pwith zeta-potential of −29.9 ± 1.4 mV, indicating nanoparticles that are stable in aqueous solution. • Highly selective calcium ionophore acts as a recognition element that extracts Ca2+ into the sensor, which causes the pH-sensitive fluorophore (chromoionophore) to deprotonate to maintain charge neutrality within the sensor with an accompanying change in fluorescence
- 14. Applications • Calcium detection assays used to dignose Bone diseases-osteoporosis Kidney disease hypercaluria Thyroid disease Hyperparathyroidism Hypertension Alzheimer’s disease