Corona green

For surface staining, cells were stained with antibodies (CD3, CD4, CD8, MHC-I, MHC-II from BD Biosciences or Biolegend, dilution 1:200) for 30–45 min in cell isolation buffer in dark tubes before analysis. For NCC function study using CoroNa Green (cell permeable, Molecular Probes), mDCTs were treated with ouabain, bumetanide and amiloride for 30 min at room temperature (RT) followed by loading CoroNa Green at a concentration of 10 μM for 1 h at RT. Cells were washed and re-suspended in PBS containing the same blockers mentioned above for 45 min and analysed in a BD Accuri C6 flow cytometer immediately. Flow cytometry data were analysed using FlowJo software.

The cellular Na⁺ content of 9-day NaCl-treated leaves was determined using the Na⁺ indicator dye, CoroNa Green (Invitrogen, United Kingdom). Sample collection and measurement was performed as previously described by Zhang et al. (2020) with some modifications. The lower epidermis of the third leaf was peeled off to expose the mesophyll cells, which were allowed to adhere to the staining solution. After staining by incubation with 20 µM CoroNa™ Green and 0.5 M mannitol for 1 hour, 20 µM FM4-64 was added for co-staining and the cells were incubated for 1 hour. The excitation wavelength of CoroNa™ Green is 488 nm and the detection wavelength ranges between 510 and 520 nm, while the detection wavelength of FM4-64 ranges between 610 and 630 nm. The concentration of Na⁺ was determined by calculating the mean intracellular fluorescence intensity of CoroNa™ Green. We set five biological repeats for each treatment group from independent plants The mean values of the observations obtained from 10 cells in each biological repeats of each treatment group represented the cellular Na⁺ concentration.

Immunofluorescent study of A549 cells was performed with cathepsin B antibody (Abcam, 1:100) and visualized by an upright microscope (Eclipse 80i, Nikon).
For the monitoring of intracellular sodium level, cells were first loaded with CoroNa Green (Invitrogen) and PI according to the manufacture's protocol. Then, cationic carriers were added and fluorescence was monitored by a fluorescent microscope. Images were recorded every 60 s. The fluorescent intensity was analyzed using an image analysis program (Image J).
To detect the release of mitochondria caused by cationic carriers, the mouse primary lung cells and A549 cells were stained with Mito-Tracker Green (Beyotime Institute of Biotechnology, Nantong, China) at a final concentration of 100 nM and Hoechst 33342 (20 μg/ml, Sigma) for 30 min. Then, cells were treated with various cationic carriers at the indicated concentration for another 2 h. The supernatant was removed gently and cells were observed under the Leica TCS SP5 confocal microscope.