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

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 express-ing only the native IP3R type 1 receptor using total internal reflection fluores-cence (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).

Automated summary

This protocol details the steps to visualize and detect Ca2+ puffs in HEK-293 cells using TIRF microscopy, offering high-speed imaging with improved signal-to-background ratio. Common pitfalls encountered while recording Ca2+ puffs using TIRF microscopy are also highlighted.