The HPTS Transport Selectivity Assay Synthetic POPC vesicles (200 nm) containing NaCl (100 mM), HEPES (10 mM), and HPTS (1 mM) were made using a similar initial method as described for the ISE-based assays. However, after the freeze-thaw cycles and twenty-five extrusions, the unencapsulated internal solution was removed via size-exclusion chromatography using G-25 Sephadex ® beads, which had first been cleaned by eluting the external solution through the column. The external solution also consisted of NaCl (100 mM) and HEPES (10 mM), and both the internal and external solutions were adjusted to pH 7. The vesicles were collected after size separation and diluted to a known concentration. During the transport experiments, the spectrophotometer excited the HPTS probe at λex = 403 nm (to excite the protonated form) and λex = 460 nm (to excite the deprotonated form), with the fluorescence emission of both being monitored at an emission wavelength of λem = 510 nm. Transport studies were first set up by adding the vesicles (0.1 mM) to a cuvette containing the external solution (2.5 mL). A base pulse of NaOH (0.5 M, 25 μL) was then added at t = -30 s to raise the pH of the external solution by ~1 unit. After confirming no vesicle leakage was occurring, the transporters were added as DMSO solutions (5 μL) at t = 0 s to initiate transport. Transport was allowed to continue until t = 230 s, where Triton X-100 (50 μL, 10%, v/v) was added to lyse the vesicles and obtain the 100% fractional fluorescence intensity (If) reading. The experiments were stopped at t = 280 s, and each experiment for each transporter concentration was repeated twice to obtain standard errors. The vesicles could also be treated with CCCP (1 mol%) before the addition of the base pulse to study transporter-mediated Cl -uniport. Vesicles could also be pretreated using BSA (1 mol%) to remove all membrane-bound fatty acids and determine the possibility of transporter-mediated H + /Cl -co-transport unaided by the fatty acid flip-flop mechanism.