If this happens, stop the experiment. After lysing the vesicles, 30 s is usually the time required to obtain a stable reading from the instrument. It should be noted that, in order to maintain the consistency of the data, it is advisable to halt all the assays at the same time. If one set of experimental data does not mimic the others, it can be due to either irregular or the absence of stirring inside the cuvette. This may cause irregular distribution of vesicles in solution, so the data from this assay is not comparable to other experimental datasets. Therefore, you should check that the stirring is on and, if possible, keep the same stirring speed throughout all assays. This can be due to several reasons, for instance, the HPTS concentration. We have observed that the optimal fluorophore concentration is 1 mM to get reproducible results reliably; the use of lower concentrations causes a decrease in the signal-to-noise ratio and, consequently, in repeatability. Also, you must ensure that all solutions have been prepared using the same conditions, namely, the same water quality, salts used, ionic strength, and temperature. This also applies to measurements (lipid and transporter concentrations in cuvettes, volumes taken from the stock or diluted solutions to perform the assays, and a consistent stirring speed during the experiments). It is advisable to do all the work in a room equipped, if possible, with air conditioning set at 25 ºC (298 K)