A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy

This protocol outlines steps to visualize and detect Ca(2+) puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP(3)) from HEK-293 cells expressing only the native IP(3)R type 1 receptor using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy offers high...

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Detalles Bibliográficos
Autores principales: Arige, Vikas, Emrich, Scott M., Yoast, Ryan E., Trebak, Mohamed, Yule, David I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225975/
https://www.ncbi.nlm.nih.gov/pubmed/34195673
http://dx.doi.org/10.1016/j.xpro.2021.100618
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author Arige, Vikas
Emrich, Scott M.
Yoast, Ryan E.
Trebak, Mohamed
Yule, David I.
author_facet Arige, Vikas
Emrich, Scott M.
Yoast, Ryan E.
Trebak, Mohamed
Yule, David I.
author_sort Arige, Vikas
collection PubMed
description This protocol outlines steps to visualize and detect Ca(2+) puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP(3)) from HEK-293 cells expressing only the native IP(3)R type 1 receptor using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy offers high axial resolution and allows imaging at high speed, with a higher signal-to-background ratio. Additionally, we shed light on commonly encountered pitfalls, which should be considered while recording Ca(2+) puffs using TIRF microscopy. For complete details on the use and execution of this protocol, please refer to Emrich et al. (2021) and Lock et al. (2015a).
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spelling pubmed-82259752021-06-29 A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy Arige, Vikas Emrich, Scott M. Yoast, Ryan E. Trebak, Mohamed Yule, David I. STAR Protoc Protocol This protocol outlines steps to visualize and detect Ca(2+) puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP(3)) from HEK-293 cells expressing only the native IP(3)R type 1 receptor using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy offers high axial resolution and allows imaging at high speed, with a higher signal-to-background ratio. Additionally, we shed light on commonly encountered pitfalls, which should be considered while recording Ca(2+) puffs using TIRF microscopy. For complete details on the use and execution of this protocol, please refer to Emrich et al. (2021) and Lock et al. (2015a). Elsevier 2021-06-17 /pmc/articles/PMC8225975/ /pubmed/34195673 http://dx.doi.org/10.1016/j.xpro.2021.100618 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Arige, Vikas
Emrich, Scott M.
Yoast, Ryan E.
Trebak, Mohamed
Yule, David I.
A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy
title A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy
title_full A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy
title_fullStr A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy
title_full_unstemmed A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy
title_short A protocol for detecting elemental calcium signals (Ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy
title_sort protocol for detecting elemental calcium signals (ca(2+) puffs) in mammalian cells using total internal reflection fluorescence microscopy
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225975/
https://www.ncbi.nlm.nih.gov/pubmed/34195673
http://dx.doi.org/10.1016/j.xpro.2021.100618
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