Hiapp

Membrane permeability was measured in standard 96-well transparent microtiter plates in a plate reader (Spectrafluor, Tecan, Salzburg, Austria). Peptides (5 μL of 0.2 mM in DMSO) were added to calcein-containing LUVs (195 μL of lipid vesicles (50 μM) in 10 mM Tris–HCl, 100 mM NaCl buffer (pH 7.4)). As positive control human islet amyloid polypeptide (hIAPP; Bachem) 5 μL of a 0.2 mM in DMSO) was added to calcein-containing LUVs. As a negative control, murine IAPP ((mIAPP; Bachem) 5 μL of 0.2 mM in DMSO) was added to calcein-containing LUVs. For blank only, 5 μL DMSO was added to calcein-containing LUVs. Directly after the addition of all components, the microtiter plate was shaken for 10 s. Fluorescence was measured from the top, every 5 min, using a 485 nm excitation filter and a 535 nm emission filter at 25 °C. The maximum leakage at the end of each measurement was determined by adding 1 μL of 10% Triton X-100 to a final concentration of 0.05% (v/v). The release of fluorescent dye was calculated according to Equation (1):
L(t) is the fraction of dye released (normalized to membrane leakage), Ft is the measured fluorescence intensity, and F0 and F100 are the fluorescence intensities at times t = 0 and after addition of Triton X-100, respectively. All membrane leakage assays were performed two times, each in duplicate, on different days, using different IAPP stock solutions.