Plan fluor 40 1.3 oil objective eclipse ti

Cells were plated in glass bottom 35 mm-petri dishes for 24 h (No. 1.5, MatTeK Corporation, United States). Intracellular Ca²⁺ labeling and imaging was described as before (Chen et al., 2019 (link)). In details, cells were labeled with 5 μm Fura-2 AM (F1221, Thermo Fisher Scientific, United States) supplied with 0.02% Pluronic F-127 (P3000MP, Thermo Fisher Scientific, United States) and then were illuminated at an alternating excitation wavelengths of 340 nm and 380 nm in an Epi-fluorescence microscope with a Plan-Fluor 40×/1.3 Oil objective (Eclipse Ti, Nikon, Japan). The emitted fluorescence was recorded at 510 nm with an Andor Zyla sCMOS camera (Oxford Instruments, United Kingdom). Exposure time was typically 100–200 ms, and images were collected every 10–20 s. Images were analyzed using MetaFluor software (Universal Imaging Corporation, United States). Fluorescent images were background corrected and cells with similar fluorescence intensity were analyzed.

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).