Abstract
In signaling cascades downstream of NK cell activating receptor engagement, Ca2+ ions are pivotal second messengers for NK cell cytotoxicity as well as cytokine production. Upon cellular activation, intracellular mobilization of Ca2+ ions initially involves depletion of endoplasmic reticulum stores, leading to subsequent Ca2+ influx through specific plasma membrane Ca2+ release activated Ca2+ channels. Multiple probes and assays for detecting intracellular Ca2+ concentrations have been developed. With the advance of multiparameter flow cytometry instrumentation, a thorough analysis of signaling in specific NK cell subsets is possible. Here, a flow cytometric method for dynamic measurements of intracellular Ca2+ concentrations in human NK cells subsets is detailed and discussed. This assay can be further adapted for specific scientific and diagnostic questions, with implications for various immunopathological conditions.
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
Similar content being viewed by others
References
Vivier E, Tomasello E, Baratin M et al (2008) Functions of natural killer cells. Nat Immunol 9:503–510
Bryceson Y, Long E (2008) Line of attack: NK cell specificity and integration of signals. Curr Opin Immunol 20:344–352
Al-Aoukaty A, Rolstad B, Giaid A et al (1998) MIP-3alpha, MIP-3beta and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells. Immunology 95:618–624
Upshaw JL, Schoon RA, Dick CJ et al (2005) The isoforms of phospholipase C-gamma are differentially used by distinct human NK activating receptors. J Immunol 175:213–218
Bryceson YT, March ME, Ljunggren H-G et al (2006) Activation, coactivation, and costimulation of resting human natural killer cells. Immunol Rev 214:73–91
Regunathan J, Chen Y, Kutlesa S et al (2006) Differential and nonredundant roles of phospholipase Cgamma2 and phospholipase Cgamma1 in the terminal maturation of NK cells. J Immunol 177:5365–5376
Caraux A, Kim N, Bell SE et al (2006) Phospholipase C-gamma2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells. Blood 107:994–1002
Tassi I, Presti R, Kim S et al (2005) Phospholipase C-gamma 2 is a critical signaling mediator for murine NK cell activating receptors. J Immunol 175:749–754
Bryceson YT, Chiang SCC, Darmanin S et al (2011) Molecular mechanisms of natural killer cell activation. J Innate Immun 3:216–226
Feske S, Gwack Y, Prakriya M et al (2006) A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441:179–185
Picard C, McCarl C-A, Papolos A et al (2009) STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity. N Engl J Med 360:1971–1980
Maul-Pavicic A, Chiang SCC, Rensing-Ehl A et al (2011) ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis. Proc Natl Acad Sci U S A 108:3324–3329
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Anasetti C, Martin PJ, June CH et al (1987) Induction of calcium flux and enhancement of cytolytic activity in natural killer cells by cross-linking of the sheep erythrocyte binding protein (CD2) and the Fc-receptor (CD16). J Immunol 139:1772–1779
Bryceson YT, March ME, Ljunggren HG et al (2006) Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion. Blood 107:159–166
Burchiel SW, Edwards BS, Kuckuck FW et al (2000) Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications. Methods 21:221–230
MacFarlane AW, Oesterling JF, Campbell KS (2010) Measuring intracellular calcium signaling in murine NK cells by flow cytometry. Methods Mol Biol 612:149–157
Di Virgilio F, Steinberg TH, Silverstein SC (1990) Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium 11:57–62
Anasetti C, Martin PJ, Morishita Y et al (1987) Human large granular lymphocytes express high affinity receptors for murine monoclonal antibodies of the IgG3 subclass. J Immunol 138:2979–2981
Bootman MD, Rietdorf K, Collins T et al (2013) Ca2+-sensitive fluorescent dyes and intracellular Ca2+ imaging. Cold Spring Harb Protoc 8:83–99
June CH, Abe R, Rabinovitch PS (2001) Measurement of intracellular calcium ions by flow cytometry. Curr Protoc Cytom Chapter 9:Unit 9.8
Acknowledgments
This work was supported by the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013)/ERC Grant Agreement no. 311335, Swedish Research Council, Swedish Foundation for Strategic Research, Swedish Cancer Foundation, Swedish Children’s Cancer Foundation, Knut and Alice Wallenberg Foundation, and the Karolinska Institute Research Foundation. J.T. is supported by a researcher internship grant provided by the Stockholm County Government and a MD/PhD fellowship provided by Karolinska Institutet.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Theorell, J., Bryceson, Y.T. (2016). Analysis of Intracellular Ca2+ Mobilization in Human NK Cell Subsets by Flow Cytometry. In: Somanchi, S. (eds) Natural Killer Cells. Methods in Molecular Biology, vol 1441. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3684-7_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3684-7_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3682-3
Online ISBN: 978-1-4939-3684-7
eBook Packages: Springer Protocols