FAVS of Pool 2 G2A Naked2-EGFP vesicles. A, purification of GFP and DiD dual-labeled G2A Naked2-associated Pool 2 vesicles is described in detail under “Experimental Procedures.” Sorting was performed on a BD Biosciences FACSAria customized with a forward scatter PMT and standardized for linearity. Unstained and single stained (GFP or DiD) vesicles were used for compensation of spectral overlap. Doubly positive DiD-counterstained EGFP-tagged Pool 2 G2A Naked2 fluorescent vesicles were gated and subjected to pulse processing analysis for doublet discrimination. Finally single doubly positive fluorescent vesicles were gated to remove GFP and DiD vesicles that were greater than 0.55 SD from their mean fluorescent intensities, respectively. Partially purified vesicles (top panel) exhibited, at best, 74% double positivity (dual fluorescence for DiD and GFP), whereas post-sorted vesicles (bottom panel) displayed greater than 99% double positivity (see “Experimental Procedures” for details). B, merged images of DiD (red) and Naked2-EGFP (green) of pre-sorted (top) and post-sorted (bottom) Pool 2 G2A Naked2-associated vesicles (bar, 10 μm). C, 5 μl of post-sorted vesicles was dried on a grid overnight at room temperature for negative staining (left panel; bar, 100 nm). For positive staining (right panel; bar, 100 nm), post-sorted vesicles were stained with 1% aqueous osmium tetroxide for 1 h. The negative staining grids or positive staining thin sections then were observed at 80 kV on a Philips FEI CM-12 electron microscope. The arrow points to a vesicle shown at higher magnification in the inset. This vesicle exhibits a single membrane bilayer. D, immunoblots showing relative abundance of Naked2, Golgin97, and Rab10 in whole cell lysates and pre-sort and post-sort Pool 2 G2A Naked2-associated vesicles. Equal amounts of protein (15 μg/lane) were loaded and separated in NuPAGE 4–12% Bis-Tris gels (Invitrogen).