Flash Photolysis of Caged IP₃ to Trigger Intercellular Ca²⁺ Waves

Abstract

Caged IP₃ is an inactive form of the second messenger IP₃, consisting of the biologically active molecule linked to a cage group through a photolabile bond. This bond is cleaved by exposure to brief “flashes” of ultraviolet (UV) light, thereby releasing the active IP₃ molecule. The protection of caged IP₃ against metabolic transformation in combination with a defined time point of fast photoliberation of IP₃ provides an efficient way to temporally and spatially control the cytosolic release of IP₃ and subsequent increase of cytoplasmic Ca²⁺. These properties make it an ideal method for kinetic studies and also a well-suited procedure to initiate intercellular Ca²⁺ waves from a point source of IP₃. This protocol describes cell loading with membrane impermeable caged IP₃ and the UV flash illumination procedure.