Nanoparticle analyzer

Blood samples were collected from the wing vein of chickens after 1 and 3 days of infection (three chickens from each group). Exosomes were extracted from the serum using Total Exosome Isolation Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Briefly, 5 mL of the blood sample was collected from infected and control chickens. The blood was incubated at room temperature (RT) for 2 h to allow clotting. Serum was isolated from the clotted blood and centrifuged at 2000 × g for 30 min at 4 °C to remove cells and debris. The supernatant was mixed with 0.2 volumes of the Total Exosome Isolation Reagent and incubated at 4 °C for 30 min. After incubation, samples were centrifuged at 10 000 × g for 10 min at RT. The supernatant was discarded, and exosomes were contained in the pellet at the bottom of the tube. Exosomes were suspended with phosphate-buffered saline (PBS; pH 7.4) and stored at ≤  −20 °C.
For characterization of exosomes, the particle size was measured using a Nanoparticle Analyzer (HORIBA, SZ-100, Kyoto, Japan). Furthermore, a Western blot assay was performed using CD81 as a exosomal marker (#56039; Cell Signaling Technology, Danvers, MA, USA) according to previously described methods [25 (link)].

For SEM, samples were deposited onto conductive glass, dried, and rinsed with diH₂O. Dry samples were coated with Au (5 nm of thickness) and observed on an S-4800 SEM. TEM observation of dry iDR-NCs samples was also conducted. Preparation of NC cross section using FIB and the following SEM observation of NC cross section was conducted on a Tescan GAIA FIB/SEM at the Advanced Imaging and Microscopy Laboratory (AIMLAB). AFM samples were casted on mica substrate, dried, and washed with diH₂O. AFM samples was observed in tapping-mode in air on a PicoForce Multimode AFM (Bruker, CA) equipped with a Nanoscope® V controller, a type E scanner head, and an OTESPA AFM cantilever. Results were analyzed using Nanoscope Software (ver. 7.3–8.15). The sizes of iDR-NCs suspended were measured using DLS on a Nanoparticle Analyzer (HORIBA Scientific, Tokyo, Japan). Bright field or fluorescence images of fluorophore-labeled iDR-NCs were taken on a Zeiss LSM 780 confocal microscope (Chesterfield, VA).