Abstract
Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Marinissen MJ, Gutkind JS (2001) G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci 22:368–376
Berridge MJ (1993) Inositol trisphosphate and calcium signalling. Nature 361:315–325
Clapham DE (1995) Calcium signaling. Cell 80:259–268
Offermanns S, Simon MI (1995) G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J Biol Chem 270:15175–15180
Ridgway EB, Ashley CC (1967) Calcium transients in single muscle fibers. Biochem Biophys Res Commun 29:229–234
Stables J, Green A, Marshall F, Fraser N, Knight E, Sautel M, Milligan G, Lee M, Rees S (1997) A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor. Anal Biochem 252:115–126
Stables J, Mattheakis LC, Chang R, Rees S (2000) Recombinant aequorin as reporter of changes in intracellular calcium in mammalian cells. Methods Enzymol 327:456–471
Brini M, Pinton P, Pozzan T, Rizzuto R (1999) Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell. Microsc Res Tech 46:380–389
Minta A, Kao JP, Tsien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264:8171–8178
Woszczek G, Chen LY, Nagineni S, Alsaaty S, Harry A, Logun C, Pawliczak R, Shelhamer JH (2007) IFN-gamma induces cysteinyl leukotriene receptor 2 expression and enhances the responsiveness of human endothelial cells to cysteinyl leukotrienes. J Immunol 178:5262–5270
Chen LY, Woszczek G, Nagineni S, Logun C, Shelhamer JH (2008) Cytosolic phospholipase A2alpha activation induced by S1P is mediated by the S1P3 receptor in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 295:L326–L335
Wilson GA, Butcher LM, Foster HR, Feber A, Roos C, Walter L, Woszczek G, Beck S, Bell CG (2014) Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases. Genome Med 6:19
Chen LY, Eberlein M, Alsaaty S, Martinez-Anton A, Barb J, Munson PJ, Danner RL, Liu Y, Logun C, Shelhamer JH, Woszczek G (2011) Cooperative and redundant signaling of leukotriene B4 and leukotriene D4 in human monocytes. Allergy 66:1304–1311
Parmentier CN, Fuerst E, McDonald J, Bowen H, Lee TH, Pease JE, Woszczek G, Cousins DJ (2012) Human T(H)2 cells respond to cysteinyl leukotrienes through selective expression of cysteinyl leukotriene receptor 1. J Allergy Clin Immunol 129:1136–1142
Woszczek G, Chen LY, Nagineni S, Shelhamer JH (2008) IL-10 inhibits cysteinyl leukotriene-induced activation of human monocytes and monocyte-derived dendritic cells. J Immunol 180:7597–7603
Foster HR, Fuerst E, Lee TH, Cousins DJ, Woszczek G (2013) Characterisation of P2Y(12) receptor responsiveness to cysteinyl leukotrienes. PLoS One 8:e58305
Foster HR, Fuerst E, Branchett W, Lee TH, Cousins DJ, Woszczek G (2016) Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor-dependent gene expression. Sci Rep 6:20461
Woszczek G, Chen LY, Nagineni S, Kern S, Barb J, Munson PJ, Logun C, Danner RL, Shelhamer JH (2008) Leukotriene D(4) induces gene expression in human monocytes through cysteinyl leukotriene type I receptor. J Allergy Clin Immunol 121:215–221
Chan V, Burgess JK, Ratoff JC, O’Connor BJ, Greenough A, Lee TH, Hirst SJ (2006) Extracellular matrix regulates enhanced eotaxin expression in asthmatic airway smooth muscle cells. Am J Respir Crit Care Med 174:379–385
Fuerst E, Foster HR, Ward JP, Corrigan CJ, Cousins DJ, Woszczek G (2014) Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells. Allergy 69:1531–1539
Kirshenbaum AS, Akin C, Wu Y, Rottem M, Goff JP, Beaven MA, Rao VK, Metcalfe DD (2003) Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI. Leuk Res 27:677–682
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Woszczek, G., Fuerst, E., Maguire, T.J.A. (2021). FLIPR Calcium Mobilization Assays in GPCR Drug Discovery. In: Martins, S.A.M., Prazeres, D.M.F. (eds) G Protein-Coupled Receptor Screening Assays. Methods in Molecular Biology, vol 2268. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1221-7_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1221-7_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1220-0
Online ISBN: 978-1-0716-1221-7
eBook Packages: Springer Protocols