Although HEPES (10 mM) is the most widely used buffer to carry out this experiment, others, such as the NaH2PO4/Na2HPO4 (10 mM) system, can be used as long as they do not interfere with the assay. The flexibility of the HPTS experiment means that different salts (MX) can be employed to prepare the inner and outer solutions. Usually, both are made up of NaCl (100 mM), but it is not uncommon to find works in the literature where M is Li + , Na + , K + , Rb + , Cs + or NMDG + and X is F -, Cl -, Br -, I -, NO3 -, AcO -, SCN -or ClO4 -. The intra-and extravesicular media composition may be dissimilar depending on the purpose of the experiment (pH, anion or dual gradient assay); however, regardless of the composition, the total ionic strength of both solutions must remain constant throughout a typical experiment.