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Calcium Signaling pp 45–82Cite as

Development and Optimization of FLIPR High Throughput Calcium Assays for Ion Channels and GPCRs

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Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 740))

Abstract

Ca2+ permeable ion channels and GPCRs linked to Ca2+ release are important drug targets, with modulation of Ca2+ signaling increasingly recognized as a valid therapeutic strategy in a range of diseases. The FLIPR is a high throughput imaging plate reader that has contributed substantially to drug discovery efforts and pharmacological characterization of receptors and ion channels coupled to Ca2+. Now in its fourth generation, the FLIPRTETRA is an industry standard for high throughput Ca2+ assays. With an increasing number of excitation LED banks and emission filter sets available; FLIPR Ca2+ assays are becoming more versatile. This chapter describes general methods for establishing robust FLIPR Ca2+ assays, incorporating practical aspects as well as suggestions for assay optimization, to guide the reader in the development and optimization of high throughput FLIPR assays for ion channels and GPCRs.

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Abbreviations

FLIPR:

Fluorescent Imaging Plate Reader

Ca2+ :

calcium ion

ATP:

adenosine triphosphate

PMCA:

Plasma Membrane Ca2+ ATPase

NCX:

Na+/Ca2+ exchanger

SERCA:

sarco/endoplasmic reticulum Ca2+ ATPase

IP3:

inositol-1,4,5,-triphosphate

RyR:

ryanodine receptors

GPCR:

G-protein coupled receptor

PIP2 :

phosphatidylinositol 4, 5 bisphosphate

DAG:

diacylglycerol

HTS:

high throughput screening

VGCC:

Voltage-gated Ca2+ channels

LGCC:

Ligand-gated Ca2+ channels

EGTA:

ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid

APTRA:

2-aminophenol-N,N,O-triacetic acid

BAPTA:

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

Kd :

dissociation constant

AM:

acetoxymethyl

ER:

endoplasmic reticulum

LED:

light-emitting diode

CCD:

charge-coupled device

PDL:

poly-D-lysine

PLL:

poly-L-lysine

PLO:

poly-L-ornithine

nAChR:

nicotinic acetylcholine receptors

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

PAR2:

protease-activated receptor 2

RFU:

relative fluorescence unit.

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  1. Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia

    Irina Vetter

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  1. , Department of Clinical Sciences and Educ, Karolinska Institutet, Tulegatan 26, Stockholm, SE-118 83, Sweden

    Md. Shahidul Islam

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Vetter, I. (2012). Development and Optimization of FLIPR High Throughput Calcium Assays for Ion Channels and GPCRs. In: Islam, M. (eds) Calcium Signaling. Advances in Experimental Medicine and Biology, vol 740. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-2888-2_3

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