The optimised assay protocol is outlined in Figure and involves the following steps: 1) Vesicles containing encapsulated lucigenin were added to each well of a 96-well plate using a multi-channel pipette (150 L per well, 2 mM POPC, internal buffer = 100 µM lucigenin in 200 mM NaNO3 with 5 mM sodium phosphate buffer, pH 7.2. External buffer = 200 mM NaNO3 with 5 mM sodium phosphate buffer, pH 7.2.). Then a solution of transporter in DMSO was added to the lipid, followed by mixing using a multi-channel pipette (3 µL of 100X stock solution). 2) Analysis was performed on a plate reader equipped with an autoinjector, which facilitated the addition of a sodium chloride spike to each well (150 µL per well, 200 mM NaCl, with 5 mM sodium phosphate buffer, pH 7.2). 3) The fluorescence emission intensity (excitation = 369 nm, emission = 503 nm) of each well was measured 5 minutes after the addition of the sodium chloride, and this was processed to give the final Hill plots. Data processing included the creation of a Stern-Volmer plot (Figure ), which was used to convert the fluorescence intensity to a chloride concentration. It is important to note that up to 20% lucigenin leakage from the vesicles may occur within two hours, which should not be confused with ion transport. Additional data calibration was performed using vesicles treated with DMSO only as a reference for 0% chloride influx and a highly potent transporter as a reference for 100% chloride influx (Equation S2).