Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane

To measure the fluorescence leakage, dilute the LUVs in 1 milliliter of HEPES Buffer 2 in a quartz fluorescence cuvette to a 100 micromolar final concentration, and add a magnetic stirrer to facilitate homogenization of the solution during the experiment.

Measure the LUVs alone during the first 100 seconds to assess the background fluorescence, before measuring the leakage as an increase in fluorescence intensity upon the addition of aliquots of peptide solution over the next 900 seconds.

To measure 100% fluorescence leakage as a positive control, add 1 microliter of Triton X-100 to the LUVs to solubilize the vesicles, resulting in a complete unquenching of the probe during the last 100 seconds of the analysis. Then, use the formula to calculate the leakage percentage at each time point.