The assay should be calibrated with the same vesicles tested in the HPTS assay if the option is available. The procedure for preparation of the standard vesicles has been used in and adapted from the journal article 'Measuring anion transport selectivity: a cautionary tale' by Xin Wu and Philip A. Gale. (1) Prepare an external solution of NaCl (100 mM) and HEPES (10 mM) as well as a NaOH base pulse solution (0.5 M) using Milli-Q water.