Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

All animal experiments were approved by the local ethics committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit Number: SHVRIAU-14-0101).

1. In Vitro Culture of Schistosomes

Note: Schistosomes represent a biohazard. Workers should wear latex gloves at all times when handling worms, schistosomal suspensions, or other related biological materials. New Zealand rabbits were infected with ~2,000 cercariae via abdominal exposure. Schistosomes at the liver stage were collected from rabbits infected with S. japonicum cercariae at 28 days post-infection by perfusion of liver and mesenteric veins. The procedures of worm collection have been referenced in previous publications reporting studies in mice¹⁸.

1. Collect parasites in a 500-mL beaker. Wash them thoroughly and gently 3x with 200 mL of preheated (37 °C) PBS (pH 7.4).
2. Separate ~200 worm pairs for each 90 mm Petri dish. Then wash the parasites thoroughly and gently 2x with 50 mL of preheated (37 °C) RPMI-1640 culture medium containing 100 U of penicillin and 100 mg/mL of streptomycin.
3. Maintain parasites in 40 mL of preheated RPMI-1640 culture medium without antibiotics at 37 °C under 5% CO₂ at a density of ~5 worm pairs /mL for 2 h in 90 mm Petri dish.
   Notes: The RPMI 1640 medium should not contain serum, otherwise exogenous extracellular vesicle will be introduced.

2. Pre-treated Condition Media

1. Collect the culture medium and then centrifuge the medium at 4 °C at 2,000 × g for 30 min to remove eggs and worm tegmental debris.
2. Transfer the supernatant to a fresh tube and then filter the solution using a 0.22 µm syringe filter.
3. Collect the filtered solution in a dialyzed bag (molecular weight cut-off: 3.5kD) and then dialyze the solution using 8% PEG8000 solution at 4 °C overnight.
   Notes: This step is important to enrich SjEVs for increasing the yield of SjEVs isolation.

3. Isolation of SjEVs

1. Transfer the dialyzed solution to a new tube and then add 0.5 volumes of the total exosome isolation reagent.
2. Mix the solution well by vortexing or pipetting up and down to ensure homogeneity of the solution.
3. Incubate the mixture at 2 °C to 8 °C overnight.
4. Centrifuge the mixture at 15,000 × g for 1 h at 2 °C to 8 °C.
5. Aspirate and discard the supernatant.
   Notes: EVs are contained in the pellet at the bottom of the tube (not visible in most of the cases).
6. Resuspend the EVs pellet in 2 mL of PBS and then store the EVs solution at -80 °C until further analysis.

4. Characterization of EVs by Western Blotting

1. Determine the protein concentration of the SjEVs sample, e.g., by BCA assay.
2. Prepare 10 – 20 µg of EVs in 22.5 µL RIPA buffer. Then add 7.5 µL of 4x loading buffer and heat for 5 min at 95 °C.
3. Separate SjEVs proteins by 12% SDS-PAGE gel.
4. Transfer the proteins to PVDF membrane and then probe with anti-CD63 antibodies (1:1,000 dilution), followed by probing with anti-rabbit IgG-HRP (1:5,000 dilution).
5. Use ECL detection reagent for membrane development and detect the signals by a chemiluminescence imaging system.

5. Characterization of EVs by Transmission Electron Microscopy

1. Add 2 µL of isolated SjEVs to 200 mesh formvar-coated grids and then allow it to dry at room temperature.
2. Stain with 2% Phosphotungstic Acid for 1 min and then allow it to dry at room temperature.
3. Load the grids onto the sample holder of the transmission electron microscope and exposed to an 80 kV electron beam for image capture.

6. SjEVs Label with PKH67 and Cell Uptake Assay

Notes: All subsequent steps were performed at ambient temperature (20 – 25 °C), unless otherwise indicated.

1. Transfer 5 µg SjEVs solution into a conical bottom polypropylene tube and adjust the volume to 12 mL by adding RPMI 1640 medium.
2. Centrifuge the SjEVs at 110,000 × g for 90 min at 4 °C to pellet SjEVs.
3. After centrifugation, carefully aspirate the supernatant.
   Note: The PKH67 ethanolic dye should not be added directly to the SjEV pellet, as this would result in heterogeneous staining and reduced cell viability.
4. Prepare a 2x EV Suspension solution by adding 1 mL of Diluent C to the SjEV pellet; to ensure complete dispersion, resuspend with gentle pipetting.
5. Just before staining in Diluent C, prepare a fresh 2x Dye Solution by adding 2 µL of the PKH67 ethanolic dye solution to 1 mL of Diluent C in a polypropylene centrifuge tube and mixing well to disperse.
6. Rapidly add the 1 mL of 2x EV Suspension (Step 6.4) to 1 mL of 2x Dye Solution (Step 6.5) and immediately mix the sample by pipetting.
7. Incubate the mixture at room temperature for 4 min.
8. Stop the staining by adding 4 mL of FBS and then incubate for 1 min.
9. Add RPMI1640 medium to adjust the volume to 12 mL and then centrifuge at 110,000 × g at 4 °C for 90 min.
10. Remove the supernatant and add RPMI1640 medium to resuspend the PKH67 labeled SjEV pellets.
11. Centrifuge at 110,000 × g at 4 °C for 90 min to wash once and then add PBS to resuspend the SjEVs pellets.

7. SjEVs Cell Uptake Assay

1. Seed mammalian cells such as NCTC clone 1469 cells or HEK293T cells in 6-well plates (2× 10⁵ cells per well) and culture the cells overnight.
2. Add 5 µg PKH67-labelled SjEVs and then culture cells at 37 °C for 2 – 4 h.
3. After incubation, remove the medium and wash the cells with PBS twice.
4. Fix the cells with 4% formaldehyde solution for 15 min and washed twice more with PBS.
5. Stain cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI).
6. Observe the cells using fluorescence microscopy.