0.8 μm filter

The primary human VS cells were cultured for 7 days. The culture medium was then replaced with a culture medium supplemented with 5% EV-depleted fetal bovine serum (FBS; Gibco #16140–071, Thermo Fisher, Waltham, MA, United States), purified by high-speed centrifugation to deplete EVs from the FBS. After 48 h, conditioned media were collected and centrifuged for 10 min at 300 g, then 10 min at 2000 g at 4°C. The supernatant was filtered through a 0.8 μm filter (Millipore; Burlington, MA, United States). EVs were pelleted by centrifugation at 100,000 g for 80 min at 4°C in a Type 70 Ti Rotor (Beckman Coulter, Brea, CA, United States).
The particle distributions (EVs) from the cells were measured using a Nanosight LM20 machine (Malvern Panalytical, Malvern, United Kingdom). A human NF2 VS-derived cell line (HEI-193) immortalized with human papilloma virus E6-7 genes was acquired from House Ear Institute (Los Angeles, CA, United States) (25 (link)) and used as a control in the particle distribution measurement (Supplementary Figure S1).

Extracellular vesicles (EVs) were isolated by ultracentrifugation and characterized using nanoparticle tracking analysis (NTA), transmission electron microscope (TEM), and western blotting (WB) for protein maker detection, as described previously [21 (link)]. Briefly, ASCs were grown to 70% confluence and then incubated in DMEM/F12 medium containing 10% exosome-depleted FBS (pre-ultracentrifuged at 120,000 g overnight). After 48 h, conditioned media was collected and centrifuged at 300 g for 10 min, then 2000g for 20 min, and then filtered through a 0.8-μm filter (Millipore), followed by centrifugation at 16,500g for 45 min to isolate ectosomes. Exosomes were isolated by subsequent centrifugation at 110,000g for 70 min. The supernatant after pelleting the EVs was collected and defined as the "EV-depleted fraction". For in vitro and in vivo experiments, EV pellets were thoroughly washed once with phosphate-buffered saline (PBS) and resuspended in PBS and then stored at -80 °C until usage. All centrifugations were performed at 4 °C.

GBM patients and healthy volunteers were recruited according to an Institutional Review Board approved protocol with informed consent. A total of 32 individuals were enroled. For the profiling study, we obtained serum samples from cancer patients (n=17), harbouring newly diagnosed or recurrent glioblastoma, as well as healthy volunteers (n=15). Cancer diagnoses were confirmed by neuropathologic examination and clinical imaging. Methylation status of MGMT in cancer patients were determined on pathologic tissues using methylation-specific PCR, performed independently by the Massachusetts General Hospital Pathology Service. For longitudinal evaluation, serial serum samples were collected from each patient (n=7) at distinctive treatment follow-ups. All serum samples were filtered through a 0.8-μm filter (Millipore) to remove cells and debris and used directly for immunomagnetic capture before mRNA analyses. All patients received standard-of-care TMZ treatment. Responder, stable and non-responder status were assigned by a neuro-oncologist (F.H.H.), without knowledge of iMER results, based on commonly used response criteria: (1) MRI evaluation for presence of gadolinium enhancement changes based on bi-dimensional accepted criteria31 (link) and (2) clinical assessment (for example, performance status, steroid dose changes and neurologic examination).

The surgical manipulations were performed on E2 embryos. A slit was made between the neural tube and right somite along the anteroposterior axis, with a sharpened tungsten needle. The embryo was then separated into a right axial structure-depleted side and a left control side. Both parts were fixed with PFA soon after the manipulation or incubated for 4 h at 38 °C on a 0.8-μm filter (Millipore) placed in a 6-cm Center-Well Culture Dish (Falcon) containing 10% fetal bovine serum/DMEM (Nissui).

Approximately 50 mg of arthropods from the STE0043 sample were washed in 70% ethanol, as previously described (Slimani et al., 2013 (link)) and crushed in 2 mL of sterile EMEM medium (Life Technologies, Saint Aubin, France) using two 3-mm tungsten beads and a TissueLyser at 25 Hz for 2 min (Qiagen, Courtaboeuf, France). The supernatant was harvested after a low speed clarification and subsequently filtered through a 0.8-μm filter (Millipore, Molsheim, France). The resulting supernatant was purified onto a discontinuous 66–30% sucrose gradient and ultracentrifuged at 130,000 g for 2 h at +4°C, as described above.
The viral fraction was harvested at the interphase between the 66 and 30% sucrose layers and fixed for 1 h at +4°C with 2% final glutaraldehyde. The fixed viral fraction was then diluted to a final volume of 4 mL in PBS, and adsorbed directly onto formvar carbon films on 400 mesh nickel grids (FCF400-Ni, EMS) by ultracentrifugation at 130,000 g for 1 h at +4°C, as previously described (Sime-Ngando et al., 1996 (link)). Grids were stained for 10 s with 1% molybdate solution in filtered water at room temperature. Electron micrographs were obtained on a Tecnai G2 transmission electron microscope (FEI) operated at 200 keV equipped with a 4096 × 4096 pixel resolution Eagle camera (FEI).