Another common method of investigating AMP-induced permeabilization is the use of efflux studies where tracer release is tracked over time. This has been done in both cell cultures , and model lipid vesicles , but generally requires large fluorescent dyes like calcein. These studies clearly show that the investigated AMPs do help the dye diffuse through the membrane, however, it is important to note that the dye release occurs on rather long time scales, on the order of 10-100 seconds. An alternative method involves trapping the pH-sensitive fluorophore pyranine in lipid vesicles and observing fluorescence changes upon ion addition . showed that ion addition to pyranine-loaded membranes increases fluorescence, due to proton exchange with externally added ions . this method alongside culture studies to show that aurein selectively permeabilizes lipid membranes for ions . Notably, the fluorescence change occurs in 10-100 seconds, despite only ions crossing the membrane, not the larger fluorophore. While release studies confirm AMP-induced permeabilization, they do not directly reveal the underlying structural and molecular mechanisms.