Chapter title |
Fluorescence Readout of a Patch Clamped Membrane by Laser Scanning Microscopy
|
---|---|
Chapter number | 23 |
Book title |
Chemotaxis
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3480-5_23 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3478-2, 978-1-4939-3480-5
|
Authors |
Matthias Gerhardt, Michael Walz, Carsten Beta, Gerhardt, Matthias, Walz, Michael, Beta, Carsten |
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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