Quantifying Membrane-Associated Fluorescence

ANS (Sigma, St. Louis, MO, USA) is a lipophilic reagent that incorporates naturally into membranes and fluoresces at 512 nm when excited at 350 nm. The ANS assay was performed in parallel on liposomes (0.86 mM phosphatidyl choline (PC), 0.24 mM phosphatidyl ethanolamine (PE) and 0.24 mM cardiolipin (CL)) and OMVs, liposomes serving as references of the ANS assay, as described earlier [23 (link)]. An increasing concentration in liposomes (1 to 5 µg/µL in Tris-KCl buffer (50 mM Tris-HCl, 200 mM KCl, pH 7.5)) as well as 1 or 2 µL of each OMV solution were incubated with 200 µL of 0.001% ANS (in Tris-KCl buffer) in a black flat-bottom 96-well plate (Corning, Corning, NY, USA). The fluorescence was measured at 512 nm after excitation at 350 nm for 100 ms using a Varioskan plate reader (Thermofisher). Concentration measurements were performed in triplicates, and the mean concentrations in lipids were calculated and used to quantify the OMVs in the experiments involving OMVs. The lipid concentration in REL606 OMVs was found to be 2 mg/mL, while that of miRFP713-OMVs was 0.7 mg/mL.