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 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).