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.

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