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A protocol for detecting elemental calcium signals (Ca²⁺ puffs) in mammalian cells using total internal reflection fluorescence microscopy.

ABSTRACT: This protocol outlines steps to visualize and detect Ca²⁺ puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP₃) from HEK-293 cells expressing only the native IP₃R 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 Ca²⁺ 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).

SUBMITTER: Arige V

PROVIDER: S-EPMC8225975 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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A protocol for detecting elemental calcium signals (Ca2+ puffs) in mammalian cells using total internal reflection fluorescence microscopy.

Arige Vikas V Emrich Scott M SM Yoast Ryan E RE Trebak Mohamed M Yule David I DI

STAR protocols 20210617 3

This protocol outlines steps to visualize and detect Ca2+ puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP3) from HEK-293 cells expressing only the native IP3R 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 co ...[more]

PMID: 34195673

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