2.3. Calcein Leakage Assay

The effect of hep 25 on the integrity of the C. albicans plasma membrane was determined by measuring the leakage of calcein from small unilamellar liposome vesicles (SUVs, 100 nm diameter). The SUVs were composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and ergosterol in a ratio of 5:4:1:2 (w/w/w/w) and were prepared as previously described [28] with some modifications. The SUVs were treated with various concentrations (0, 6.25, 12.5, 25 and 50 μg/mL) of hep 25 at room temperature (RT). To evaluate pore formation and membrane integrity, a Cary Eclipse Fluorescence Spectrophotometer (PerkinElmer, Santa Clara, CA, USA) was used to measure the leakage of the calcein dye at excitation (λex) and emission (λem) of 490 and 535 nm, respectively. The percentage of dye-leakage was calculated as follows: % dye leakage = 100 × [(F − F0)/(Ft − F0)], where F is the fluorescence intensity after the peptide treatment, Ft is the fluorescence intensity with Triton X-100, and F0 is the fluorescence intensity of the untreated control SUVs. PC, PE and PI were purchased from Avanti Polar Lipids (Alabaster, AL, USA).