ER Calcium Dynamics Measurement by FRET

Cells were plated in glass bottom 35 mm-petri dishes for 24 h (No. 1.5, MatTeK Corporation, United States) and transiently transfected with the fluorescence resonance energy transfer (FRET)-based D1ER cameleon (Palmer et al., 2004 (link)). FRET imaging was described as in the previous report (Chen et al., 2019 (link)). Briefly, cells were imaged by an Epi-fluorescence microscope with a Plan-Fluor 40×/1.3 Oil objective (Eclipse Ti, Nikon, Japan). The emission ratio of the cameleon was accomplished by 425 nm excitation wavelength with a dichroic mirror 515 nm and two emission filters (475 nm for ECFP and 535 nm for citrine-YFP) (Chroma Technology Corporation, United States). Changes in ER Ca²⁺ were expressed as the FRET-to-CFP emission ratio. Images were analyzed using MetaFluor software (Universal Imaging Corporation, United States).