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 |
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United States
01.01.2016
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Abstract | 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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AbstractList | 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. |
Author | Walz, Michael Gerhardt, Matthias Beta, Carsten |
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Keywords | Dictyostelium discoideum Patch clamp Confocal microscopy Chemotaxis Actin dynamics Excitable dynamics |
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SubjectTerms | Actins - metabolism Cell Membrane - metabolism Cells, Cultured Chemotaxis Dictyostelium - physiology Microscopy, Confocal - methods Patch-Clamp Techniques |
Title | Fluorescence Readout of a Patch Clamped Membrane by Laser Scanning Microscopy |
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