-When the initial drop of unprocessed data is larger than 20-30% of the total intensity (5-10% is normal), too much lucigenin is present outside of the liposomes. This might be caused by a poor separation during gel filtration, or by lucigenin leaking out of the liposomes over time (see next point). In the first case, the collection of liposomes from the size exclusion column should be stopped earlier, or a longer column should be used. Alternatively, two commercial pre-packed columns can be placed in series one above the other. We use this strategy when working in Na2SO4 instead of NaNO3 (see Section 4.5) or with SPBA instead of lucigenin as a probe (see Section 4.1).