Dg 5 wavelength switcher

Cells grown on poly-l-lysine–coated coverslips were loaded with 2 μM Fura-2 AM and 0.02% pluronic F-127 for 30 min at 37°C in isotonic solution containing 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.9 mM MgCl₂,10 mM glucose, and 10 mM Hepes (pH 7.4 by NaOH). The same solution but without CaCl₂ was used as imaging buffer. Cells were then washed twice with imaging buffer and transferred into the imaging chamber. GPN (600 μM) was applied to the cells to induce Ca²⁺ release from lysosomes. TG (2 μM) was applied to the cells to induce Ca²⁺ release from endoplasmic reticulum. Calcium transients were captured using a calcium imaging system consisting of a DG-5 wavelength switcher (Sutter Instrument), an ORCA-Flash4.0 LT+ complementary metal oxide-semiconductor (CMOS) camera (Hamamatsu), and a Ti2 microscope (Nikon). Data were collected and analyzed using MetaFluor software (Molecular Devices). Ratiometric measurements were performed by switching the excitation wavelength from 340 to 380 nm and quantifying emission at 510 ± 40 nm.

EYFP-H148Q/I152L was used to monitor changes in intracellular iodide concentrations. The fluorescent intensity of this YFP variant decreases with increasing iodide concentrations. HEK293T cells were cotransfected with EYFP-H148Q/I152L and the indicated plasmid at 36 hours before imaging and were plated onto poly-l-lysine–coated coverslips. Cells were washed with PBS and monitored using an imaging system consisting of a DG-5 wavelength switcher (Sutter Instrument), an ORCA-Flash4.0 LT+ CMOS camera (Hamamatsu), and a Ti2 microscope (Nikon). The imaging buffer contained 140 mM NaCl, 3 mM KCl, 2 mM K₂HPO₄, 1 mM CaCl₂, 1 mM MgSO₄, and 5 mM Hepes (pH of 7.4). The fluorescent intensities were quantified with MetaFluor software (Molecular Devices).

SH-SY5Y cells and hippocampal neurons were used for calcium imaging. SH-SY5Y cells were incubated with 10 μM all-trans retinoic acid for 24 h before experiments. Cells were loaded with 1 μM Fura2-AM dye (Thermo Fisher Scientific) for 30 min at 37°C and washed twice using Tyrode’s solution. ABC294640 (30, 60, or 90 μM) and ionomycin (1 μM) were added at the indicated time. Calcium transients were captured by live-cell imaging using a calcium imaging system consisting of a DG-5 wavelength switcher (Sutter Instrument), an ORCA-Flash4.0 LT+ CMOS camera (Hamamatsu), and a Ti2 microscope (Nikon).