-One of the disadvantages of the lucigenin assay is the intrinsic ability of lucigenin to permeate through lipid membranes. Therefore, once the external lucigenin is removed from the liposomes using gel filtration, a concentration gradient of lucigenin is present and the lucigenin starts to slowly leak from the liposomes into the external solution. Due to this effect, each prepared batch of liposomes has limited-shelf life, which can be extended a bit by storing the suspension in the fridge (not the freezer). Practically, the leaking of lucigenin can result in an increasing initial drop of fluorescence intensity over time in the individual transport experiments (see Figure ), which could be misinterpreted to be very fast transport. Therefore, the prepared batch of liposomes should be used as soon as possible (within 2-3 h).