Egg pc

The quenching assay based on a previous report (Athar et al., 2004 (link)). 180 nmol of PC (eggPC from Sigma–Aldrich, DPPC, POPC, PLPC, PAPC, and PDPC from Avanti) and 5.6 nmol of NBD-TG (Setareh Biotech, LLC, Eugene, OR) were mixed and dried in glass vials under a stream of nitrogen gas. Then, drying was completed in a centrifugal evaporator for 15 min. The dried lipid film was resuspended in 400 μl assay buffer (10 mM Tris-HCl (pH7.4), 150 mM NaCl, and 2 mM EDTA), freeze-thawed five times, and then filtered 21 times through a 50 nm pore size polycarbonate membrane (Nucleopore Track-Etch Membrane, GE Healthcare) using a mini-extruder (Avanti). The solution was centrifuged at 16,100×g (18°C, 10 min), and the supernatant was used as PC/NBD-TG vesicles. Fluorescence of vesicles was measured with a plate reader (ARVO X3) using 485 nm excitation and 535 nm emission wavelengths. Total fluorescence was determined by adding 97 μl of isopropanol to 3 μl of vesicles. Quenching of NBD-TG was calculated as follows: % quenching = (total fluorescence—fluorescence of vesicles)/total fluorescence × 100. We noted that the TG to PC ratio largely affects the quenching efficiency, thus the optimal concentration (which is around 3% TG in our experience) might vary between vials.

Powder form of Egg-PC (Sigma Aldrich) and cholesterol (Sigma Aldrich) was dissolved in chloroform. Next, 250 μM chloroform–solubilized PC was mixed with 250 μM chloroform–solubilized cholesterol and kept for overnight evaporation. The thin lipid film of mixed lipids obtained after removal of residual chloroform was dissolved in 500 μl of 20 mM Tris pH 7.5 and 150 mM NaCl with gentle pipetting. The solution was next incubated at 55 °C for 30 min. This colloidal heated solution was subjected to extrusion as described above. The PC-Chol unilamellar vesicles were used for downstream experiments and the remaining was stored at 4 °C for not more than 3 days.