Corona green am

At 72 hpf, embryos were stained with a fluorescent sodium indicator dye (CoroNa Green AM, Invitrogen, Waltham, MA, USA) to quantify embryonic sodium concentrations in situ. At 72 hpf, the embryos were rinsed three times with reverse osmosis (RO) water and then placed in a working solution containing RO water, 20% Pluronic F-127 (Invitrogen, Waltham, MA, USA), and 10 μM CoroNa Green AM for 1.5 h. The working solution was then aspirated, and embryos were washed with RO water three times for 5 min each. Finally, embryos were immobilized with 100 mg/L MS-222 for 3 min, transferred into 96-well plates, and imaged under transmitted light and a FITC filter on our ImageXpress Micro XLS Widefield High-Content Screening System within MetaXpress 6.0.3.1658 (Molecular Devices, Sunnyvale, CA, USA). Body length, pericardial area, and yolk sac area were manually quantified within MetaXpress using images captured under transmitted light, whereas total area of sodium-derived fluorescence in the head, trunk and yolk sac was quantified with a custom module within MetaXpress using images captured under a FITC filter.

BT-549, Hs578T, and HCC1806 cells were plated on glass coverslips, grown to 60–80% confluency and loaded with 2.5 μM Cal-520 AM (ATT Bioquest, Sunnyvale, CA, USA) or 5 μM CoroNA Green AM (ThermoFisher Scientific) for imaging of intracellular Ca²⁺ and Na⁺, respectively. Cell culture media was supplemented with 0.2% Pluronic F-127 (Biotium, Fremont, CA, USA) to facilitate dye loading. Cells were incubated with dye solution for 30 min at 37°C, washed with Hank’s Balanced Salt Solution (HBSS), and incubated at room temperature for 15 min in the dark. Dye-loaded cells were stored at 37 °C if imaged later than 1 hr after dye loading. Unless otherwise indicated, all cells were imaged in standard HBSS with 1.8 mM Ca²⁺ within 4 hr of dye loading. To investigate TRPC1/4/5 activity, cells were pretreated, after dye loading, for 5 min with 10 nM Pico145. Imaging was performed with a Nikon Eclipse TE2000-U inverted microscope with a 20x objective. Images were acquired with an Andor iXon camera and evaluated using Metamorph software. Fluorescent intensity was analyzed cell-by-cell using ImageJ software with personnel blinded to experimental conditions. Fluorescence intensity data are represented as corrected total cell fluorescence (CTCF) = Integrated density - (cell area X mean background fluorescence).