Rabbit anti-human actin (Sigma-Aldrich, A2066) used at 1:2000; mouse monoclonal anti-human Annexin A2 (BD Biosciences, 610069) used at 1:1000; mouse monoclonal anti-human CD63 (ThermoFisher Scientific, 10628D) used at 1:500. Q-27 mouse monoclonal anti-human GGA1 (Santa Cruz Biotechnology, sc-101257) used at 1:500. Secondary antibodies goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2004) and goat anti-mouse IgG (Santa Cruz Biotechnology, sc2005) were used at 1:2000. All antibodies were diluted in phosphate buffered saline (PBS).
Cell lines and cell culture
Human pancreatic cancer cell line PANC1 was maintained in DMEM (Sigma-Aldrich, D6546) containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 1 mM penicillin and 1 mM streptomycin. Mouse neural stem cells (NSCs) were cultured as previously described.
Extracellular vesicle isolation
PANC1 cells were grown to 70% confluence in a T75 flask at which point the medium was changed to 10 ml serum-free DMEM and incubated for 24 h. EVs were collected by ultracentrifugation as previously described. Briefly, the medium was collected and sequentially centrifuged at 300 g for 15 min, 1,000 g for 15 min, 100,000 g for 90 min. The EV pellet was washed in serum-free DMEM without phenol red (SF-DMEM; GIBCO, 21063) centrifuged at 100,000 g for 30–60 min. EVs were resuspended in SF-DMEM at 10 µl per 10 ml starting material. EVs from NSCs were also produced as previously described. Briefly, 12 million cells were seeded per T75 flask and incubated overnight. The medium was collected and EVs isolated as described above.
Cells were incubated in an appropriated volume of ice-cold lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 1 mM EDTA, 1% triton X-100, pH 6.8) at 4°C for 10 min. The lysate was collected and insoluble material pelleted at 10,000 g for 10 min at 4°C. The supernatant was collected and sample buffer added (final: 50 mM Tris-HCl, 2% (w/v) sodium dodecyl sulphate (SDS), 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol, 100 mM DTT, pH 6.8). Samples were boiled for 10 min and stored at -20°C.
Tunable resistance pulse sensing (TRPS)
TRPS were measured using the qNano (Izon Science, UK). The polyurethane nanopore NP100 (Izon Science, part A33255) was used for all measurements and was axially stretched to 46.99 mm. EV samples were diluted in PBS as required and 40 µl loaded into the instrument. Measurement time was up to 2 min depending on the instrument stability. The system was calibrated with 200 nm polystyrene beads diluted in PBS. Data analysis was carried out with Izon Control Suite software (Izon Science).
Acetylcholine esterase activity assay
10 µl EVs were lysed in 0.5% (v/v) Triton X-100 in PBS and assayed for acetylcholine esterase activity with a colorimetric assay kit from Abcam (ab138871) as per the manufacturer's instructions. The absorbance was measured at 410 nm over time and plotted against a buffer alone control.
EVs (10–20 µl per treatment) were resuspended in 100 µl of either SF-DMEM or versene solution (GIBCO, 15040-066) containing 0.48 mM EDTA and incubated at 37°C for 30 min. EVs were pelleted at 100,000 g for 30 min. The supernatant was collected and the pellet resuspended in 100 µl SF-DMEM. All samples were mixed with sample buffer (as above) and boiled for 10 min. Samples were analysed by western blotting.
All samples were resolved by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes for blotting. Membranes were blocked with 0.05% (w/v) skim milk powder in PBS containing 0.1% Tween-20 (PBS-Tween) for 30 min at room temperature. Membranes were then probed with an appropriate dilution of primary antibody overnight at 4°C. Membranes were washed three times in PBS-Tween before incubation in diluted secondary antibody for 1 h at room temperature. Membranes were washed as before and developed with ECL (Cyanagen, Westar XLS100) using a Bio-Rad Chemi Doc XRS system. Membranes were stripped with Restore plus (ThermoFisher Scientific, 46430) as per manufacturer's instructions.
Mass spectrometry analysis
Samples prepared as described above for EDTA treatment and submitted for mass spectrometry analysis using Thermo Orbitrap Q Exactive with EASY-spray source and Dionex RSLC 3000 UPLC. For graphical representation, the following equation (ratio = (EDTA sup/EDTA pellet)+1 / (untreated sup/untreated pellet)+1) was used to obtain a ratio of protein detected in the supernatant upon EDTA treatment with respect to the untreated control.
EVs (10–20 µl per treatment) were resuspended in 100 µl of either SF-DMEM, trypsin solution (Sigma-Aldrich, 4674) diluted to 2.5 mg/ml SF-DMEM, 0.5% Triton X-100 (Sigma-Aldrich, T8787) diluted in SF-DMEM or a combination of both trypsin and Triton X-100. EVs were incubated at 37°C for 30 min. They were then pelleted at 100,000 g for 30 min. The supernatant was collected and pellet resuspended in 100 µl SF-DMEM. Samples were mixed with sample buffer (as described above), boiled for 10 min and analysed by western blotting.