A lipid mixture composed of POPC:DOPE:Cholesterol:GD1a:Biotin-DPPE:TR-DHPE by molar ratio of 37.4:20:40:2:0.5:0.1 dissolved in organic solvents (chloroform and methanol) was taken in a glass vial. The solvents were removed under a flow of argon gas, and the film was dried under house vacuum for at least 12 h. The hydration solution containing a nucleic acid-binding dye is added to the vial and the film is hydrated by vortexing. The dispersion is transferred to a 0.6 mL tube and then freeze-thawed 5 times. The dispersion was next extruded through 100 nm or 200 nm polycarbonate filters using a mini extruder device (Avanti Polar Lipids) at least 31 times. The vesicles were purified on a small size-exclusion column packed with Sepharose CL-4B gel (0.8-1 mL wet volume). Eluent was collected in small fractions (70-90 µL) in 0.2 mL tubes. The fractions containing vesicles were identified from Texas Red-DHPE fluorescence by checking the tubes under UV lamp. Vesicle suspensions were stored at 4 °C and typically used within 2 months. We found that vesicles encapsulating QuantiFluor were stable for over 1 year when stored at 4 °C and can be used successfully for the content transfer experiments. We measured the size distribution and polydispersity of the vesicles using dynamic light scattering using a NanoBrook Omni particle size and zeta potential analyzer (Brookhaven Instruments. For typical vesicle preparations, we measured effective diameters (deff) of 124.8±2.9 nm (polydispersity = 0.061) and 160.0±3.4 nm (polydispersity = 0.274) for vesicles generated by extrusion through 100 nm and 200 nm membrane filters respectively (Figure ). For simplicity, we refer to the above two kinds of vesicles as 100 nm and 200 nm vesicles or unilamellar vesicles (ULVs). The 100 nm vesicles were imaged by cryo-TEM to check for unilamellarity (Figure ). Most vesicles were found to be unilamellar with some showing multi-compartment (vesicle-invesicle) architecture.