Liposome Scrambling and Leakage Assays

For the scrambling assay^{43 (link)}, NBD-labeled liposomes were prepared by adding 1% (w/v) nitrobenzoxadiazole (NBD)-labeled lipid (NBD-PE) to the lipid mixture. The scrambling assay was performed in a 100 μl quartz cuvette at room temperature (250 µM final lipid concentration). The NBD-labeled liposomes were incubated with CLIC5 (25 μM), and the NBD fluorescence was monitored before and following the addition of dithionite (to 5 mM) using 460/538 nm excitation/emission, respectively (Jasco FP-8500 spectrofluorometer). Additional dithionite (5 mM) and Triton X-100 (0.5% w/v) were added to dissolve the liposomes and allow complete quenching of the NBD fluorescence.
For the NBD-glucose leakage control assay^{43 (link)}, the lipid film was equilibrated with NBD-glucose-containing buffer (12.6 µM). Excess NBD-glucose was removed by passing the liposomes through a desalting column (PD MiniTrap G25, Cytiva) pre-washed with NBD-glucose-free buffer. Next, the experimental procedure was performed similarly to the scrambling assay, with first dithionite addition (to 1 mM), following second dithionite addition and (1 mM) and Triton X-100 (0.5% w/v) to quench the NBD-glucose signal completely.