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

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).