Protocol for measuring labile cytosolic Zn²⁺ using an in situ calibration of a genetically encoded FRET sensor

Zinc (Zn²⁺) plays roles in structure, catalysis, and signaling. The majority of cellular Zn²⁺ is bound by proteins, but a fraction of total Zn²⁺ exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn²⁺ using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn²⁺ solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn²⁺ concentration in single cells. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al.¹.