Abstract
The measurement and manipulation of cytosolic free Ca2+ of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca2+ signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils, and are discussed here, especially the use of fluorescent indicators of Ca2+ and photoactivation of reagents involved in Ca2+ signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we outline some of the techniques that have been used to monitor, visualize, and manipulate Ca2+ in neutrophils.
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Hallett MB, Hodges R, Cadman M et al (1999) Techniques for measuring and manipulating free Ca2+ in the cytosol and organelles of neutrophils. J Immunol Methods 232:77–88
Tepikin AV (2000) Calcium signalling: a practical approach, 2nd edn. Oxford Univ. Press, Oxford, U.K, p 230
Pozzan T, Lew DP, Wollheim CB et al (1983) Is cytosolic ionized calcium regulating neutrophil activation? Science 221:1413–1415
Hallett MB, Davies EV, Pettit EJ (1996) Fluorescent methods for measuring and imaging the cytosolic free Ca2+ in neutrophils. Methods 9:591–606
Scanlon M, Williams DA, Fay FS (1987) A Ca2+-insensitive form of fura2 associated with polymorphonuclear leukocytes-assessment and accurate Ca2+ measurement. J Biol Chem 262:6308–6312
Al-Mohanna FA, Hallett MB (1988) The use of fura 2 to determine the relationship between intracellular free Ca2+ and oxidase activation in rat neutrophils. Cell Calcium 8:17–26
Laffafian I, Hallett MB (1998) Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells. Biophys J 75:2558–2563
Laffafian I, Hallett MB (2000) Gentle micro-injection for myeloid cells using SLAM. Blood 95:3270–3271
Von Tscharner V, Deranleau DA, Baggiolini M (1986) Calcium fluxes and calcium buffering in human neutrophils. J Biol Chem 261:10163–10168
Pettit EJ, Hallett MB (1996) Localized and global cytosolic Ca2+ changes in neutrophils during engagement of CD11b/CD18 integrin visualized using confocal laser scanning reconstruction. J Cell Sci 109:1689–1694
Dewitt S, Darley R, Hallett MB (2009) Translocation or just location? Pseudopodia affect fluorescent signals. J Cell Biol 184:197–203
Dewitt S, Hallett MB (2011) Optical complexities of living cytoplasm—implications for live cell imaging and photo-micromanipulation techniques. J Microsc 241:221–224
Hallett M, Dewitt S (2011) A trick of the light: the optical properties of living cytoplasm which can mislead. Integr Biol 3:180–184
Hillson EJ, Hallett MB (2007) Localised and rapid Ca2+ micro-events in human neutrophils: conventional Ca2+ puffs and global waves without peripheral-restriction or wave cycling. Cell Calcium 41:525–536
Pettit EJ, Hallett MB (1995) Early Ca2+ signalling events in neutrophils detected by rapid confocal laser scanning. Biochem J 310:445–448
Davies EV, Hallett MB (1996) Near membrane Ca2+ changes resulting from store release in neutrophils: detection by FFP-18. Cell Calcium 19:355–362
Davies EV, Hallett MB (1998) High micromolar Ca2+ beneath the plasma membrane in stimulated neutrophils. Biochem Biophys Res Commun 248:679–683
Sun XR, Badura A, Pacheco DA et al (2013) Fast GCaMPs for improved tracking of neuronal activity. Nat Commun 4:2170
Suzuki J, Kanemaru K, Ishii K et al (2014) Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat Commun 5:4153
Roberts RE, Vervliet T, Bultynck G et al (2017) Dynamics of ezrin location at the plasma membrane: relevance to neutrophil spreading. Eur J Clin Investig 47:148
Roberts RE (2017) The μ-calpain-ezrin axis: A potential target for therapy in inflammatory disease. PhD Thesis: Cardiff University. http://orca.cf.ac.uk/id/eprint/108477
McDonald JU, Cortini A, Rosas M et al (2011) In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils. FASEB J 25:1972–1982
Pettit EJ, Hallett MB (1998) Release of “caged” cytosolic Ca2+ triggers rapid spreading of human neutrophils adherent via integrin engagement. J Cell Sci 111:2209–2215
Ellis-Davies GCR (2007) Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat Methods 4:619–628
Brasen JC, Dewitt S, Hallett MB (2010) A reporter of UV intensity delivered to the cytosol during photolytic uncaging. Biophys J 98:L25–L27
Armitage P, Berry G (1987) Statistical methods in medical research, 2nd edn. Blackwell Scientific, Boston, MA, pp 181–182
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Hallett, M.B., Roberts, R.E., Dewitt, S. (2020). Optical Methods for the Measurement and Manipulation of Cytosolic Calcium Signals in Neutrophils. In: Quinn, M., DeLeo, F. (eds) Neutrophil. Methods in Molecular Biology, vol 2087. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0154-9_14
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DOI: https://doi.org/10.1007/978-1-0716-0154-9_14
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