Fei tecnai 20

Tf-Au NPs and PEG-Au NPs were synthesized using a citrate reduction method and functionalized with the appropriate ligands following the protocols shown in Figure 1. PEG-Au NPs were synthesized by adsorbing 5 kDa methoxy-terminated PEG molecules onto the surface of 15 nm Au NPs through the interaction with their terminal thiol groups. For Tf-Au NP synthesis, transferrin protein was first conjugated to a Cy5 fluorophore, then to an amine-reactive 5 kDa PEG spacer with a terminal protected orthopyridyl disulfide (OPSS) thiol group. This construct was then adsorbed onto Au NP surfaces through the formation of a thiol-gold bond. The surface was further stabilized by backfilling it with a thiol-terminated 2 kDa methoxy PEG. Absorbance and fluorescence profiles of both Au NPs were measured using a UV-1601PC UV Vis (Mandel, Guelph, Canada) and a FluoroMax-3 fluorometer (Horiba, Kyoto, Japan), respectively. Electron microscopy was performed using a FEI Tecnai 20 (Thermo Fisher Scientific, Waltham, MA). The hydrodynamic diameter (D_H), polydispersity index (PdI), and zeta potential (ζ) of each suspension were measured using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK).

The EM grids were mounted on to a room temperature-equilibrated holder and subsequently introduced into a FEI Tecnai 20 transmission electron microscope (FEI Eindhoven Holland). Images (2048 pixels × 2048 pixels) were acquired using a US1000 camera (Gatan) at 50000× (nominal magnification, the corresponding pixel size was 0.21 nm). OMVs were found in gradient fractions where OmpA was detected by Western-blot, demonstrating that the OmpA screening is a suitable method for identifying OMV-enriched iodixanol gradient fractions (see Supplementary Figure S2).

A TEM sample was prepared with minor modifications from the previous study (Golding et al., 2016 (link)). The sample was adsorbed for 1 min to a formvar film on a carbon-coated 400-mesh copper grid. It was then washed 3 times in distilled water and negatively contrasted with 2% methylamine tungstate (Nano-W; Nanoprobes, Yaphank, NY, United States). The image was taken at 200 kV using a FEI Tecnai 20 transmission electron microscope (FEI Company, Hillsboro, OR, United States). The magnifications of 3,500× to 19,000× was considered for TEM images.

Fluid samples for transmission electron microscopy were fixed in 2% glutaraldehyde/1% paraformaldehyde. Samples were adsorbed to glow discharged carbon-coated formvar films on a 400-mesh copper grids for 1 min, and negatively stained with 2% methylamine tungstate (Nano- W; Nanoprobes, Yaphank, NY, USA). Specimens were observed at 200 kV in an FEI Tecnai 20 transmission electron microscope (FEI Company, Hillsboro, OR, USA) operated, and at instrument magnifications of X25,500 to X71,000. Digital images of the specimens were acquired using an AMT Advantage XR 12 CCD camera (AMT, Danvers, MA, USA).