Fluorescence Readout of a Patch Clamped Membrane by Laser Scanning Microscopy
In this chapter, we describe how to shield a patch of a cell membrane against extracellularly applied chemoattractant stimuli. Classical patch clamp methodology is applied to allow for controlled shielding of a membrane patch by measuring the seal resistivity. In Dictyostelium cells, a seal resistiv...
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Published in | Methods in molecular biology (Clifton, N.J.) Vol. 1407; p. 325 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
01.01.2016
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Subjects | |
Online Access | Get more information |
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Summary: | In this chapter, we describe how to shield a patch of a cell membrane against extracellularly applied chemoattractant stimuli. Classical patch clamp methodology is applied to allow for controlled shielding of a membrane patch by measuring the seal resistivity. In Dictyostelium cells, a seal resistivity of 50 MΩ proved to be tight enough to exclude molecules from diffusing into the shielded membrane region. This allowed for separating a shielded and a non-shielded region of a cell membrane to study the spatiotemporal dynamics of intracellular chemotactic signaling events at the interface between shielded and non-shielded areas. The spatiotemporal dynamics of signaling events in the membrane was read out by means of appropriate fluorescent markers using laser scanning confocal microscopy. |
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ISSN: | 1940-6029 |
DOI: | 10.1007/978-1-4939-3480-5_23 |