The diffusion and transport of carboxylates into liposomes was studied by fluoresce spectroscopy using lucigenin and HPTS as probes, and by a chloride ion-selective electrode (Cl-ISE). These are the most common methods for monitoring the transmembrane transport of carboxylates. All the experiments were carried out by using liposomes (200 nm) prepared from a mixture of 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (ratio = 7:3) following standard methods. The total lipid concentration used for fluorescence and Cl-ISE experiments were 0.4 mM and 1 mM, respectively. Sodium carboxylate salts were prepared by reacting the corresponding carboxylic acid (1 equivalent) and sodium hydroxide (1 equivalent for monocarboxylates and 2 equivalents for dicarboxylates) in milli-Q water to get a 1 M concentration (Figure and S2), while sodium acetate was acquired commercially. For sodium naphthoate, sodium 6-hydroxy-2-naphthoate, sodium terephthalate, sodium 2,4-dihydroxybenzoate and sodium 2,4,6-trigydroxybenzoate the concentration was reduced to 0.5 M due to their limited solubility in water. It should be noted that carboxylates were prepared in milli-Q water instead of a buffer solution because many of the carboxylates used in this study showed higher solubility in pure water. However, pH measurements were taken before and after each experiment to ensure that the buffer solutions used in each method prevented significant changes in the pH upon addition of the carboxylate solutions (Table ). For the lucigenin assay, the lipid film was hydrated with a solution of 0.8 mM lucigenin (10,10'dimethyl-9,9'-biacridinium nitrate), 225 mM NaNO3, 5 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1yl]ethane-1-sulfonic acid), pH 7. In the HPTS assay, lipid film was hydrated with 0.1 mM HPTS (8hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) in 225 mM NaNO3, 5 mM HEPES, pH 7 or 0.1 mM HPTS in 112.5 mM Na2SO4, 5 mM HEPES. For the Cl-ISE assay, the lipid film was hydrated with a solution of 488 mM NaCl, 5 mM HEPES, pH 7, while an external solution of 225 mM Na2SO4, 5 mM HEPES, pH 7 was used. For additional details see the electronic supporting information. Before conducting the fluorescence experiments, we evaluated the effect of the various aliphatic and aromatic carboxylates on the lucigenin emission. For that, 25 mM of the corresponding carboxylate was added to 0.8 mM lucigenin in 225 mM NaNO3, 5 mM HEPES, pH 7. These conditions were selected as they are standard buffer solutions for studying anion transport. The carboxylate pulse reduced the lucigenin fluorescence intensity at 505 nm to 31-75%, depending on the carboxylate added (Figure ). Moreover, aromatic carboxylates showed more quenching of the lucigenin fluorescence intensity. Additionally, dicarboxylates did not show significant differences compared to their monocarboxylate analogues. For comparison, a solution of 25 mM chloride (Cl -) was also tested, as it is a well-known lucigenin quencher.