Essential functions of neutrophils, including chemotaxis and phagocytosis, are directed in part by phospholipid signaling. Detailed elucidation of these pathways was hampered by the paucity of methods to study phospholipid localization and dynamics. The development of genetically encoded lipid-specific probes circumvented this limitation. The probes are chimeric constructs consisting of a specific lipid-binding domain fused to a fluorescent protein. This chapter describes a protocol to transiently transfect primary murine neutrophils with such probes in order to localize phospholipids in live cells, and provides a compendium of the types of lipid-binding domains currently used to visualize phospholipids.
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The authors wish to acknowledge the support of Dr. Michael Glogauer in the establishment of the above-described transfection protocol. B.E.S. is supported by the Department of Anesthesia at the University of Toronto. M.A.O.M. is supported by the Dental Research Institute (DRI) grant, Javenthey Soobiah Scholarship and the Heidi Sternbach Scholarship. S. G. is the current holder of the Pitblado Chair in Cell Biology; research in the authors’ laboratory is supported by Cystic Fibrosis Canada and by the Canadian Institutes of Health Research.
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