Live-cell imaging of Aqp2-GFP reporter cells (Vehicle, 72hrs)

Live-cell imaging of Aqp2-GFP reporter cells was performed using a Zeiss LSM 980 confocal microscope (Carl Zeiss, Germany). Cells were cultured on Transwell inserts to support appropriate Aqp2 expression and imaged using a 10×/0.30 NA objective lens. The long working distance of this objective enabled imaging through the Transwell system. GFP fluorescence was excited using the appropriate laser line and detected using standard GFP acquisition settings. Time-lapse imaging was conducted over a 72-hour period, with images acquired every hour. To maximize signal detection, the confocal pinhole was opened to 15.88 Airy units (corresponding to a pinhole diameter of 708 µm). During imaging, cells were maintained in serum-free DMEM/F-12 medium supplemented with HEPES and lacking phenol red to minimize background fluorescence. The microscope was equipped with a temperature-controlled environmental chamber maintained at 37 °C to ensure stable physiological conditions throughout the imaging period.