Mouse anti cd9

Immuno-EM analysis of whole-mount BMMSC-EVs was performed as previously described 26 . BMMSC-EVs were isolated by differential centrifugation followed by sucrose density gradient purification. Fractions corresponding to the densities 1.072-1.0899 g/mL, 1.1082-1.972 g/mL and 1.2025-1.2575 g/mL were pooled and pelleted by centrifugation at 100,000 × g for 16 h. The pellets of purified EVs were fixed in 2% paraformaldehyde and mounted on formvar/carbon-coated TEM grids. EVs were labelled with mouse anti-CD9 (1:100; Biolegend) or mouse anti-human CD63 (1:300; DSHB Hybridoma Product H5C6, deposited by August, J.T. / Hildreth, J.E.K.) followed by rabbit anti-mouse (1:200, Rockland, 610-4120) and Protein A coupled to 10 nm colloidal gold (1:50, CMC, UMC Utrecht), contrasted with uranyl oxalate (pH 7), and then contrasted-embedded in a mixture of 2% methyl cellulose/4% uranyl acetate (pH 4). Grids were imaged at 80 kV with a Tecnai T-12 Transmission Electron Microscope (FEI). Size of individual vesicles was measured in at least 5 different frames using FIJI 27 (link).

EV and CMM12 cell pellets were lysed in respectively 100 and 150 μl lysis buffer containing 0.625 M Tris/HCl (pH 6.8), 2.5% SDS and 10% glycerol. The cell lysate was depleted of nuclei by centrifugation (13 000 rpm for 1 min) and collection of the supernatant. EV and cell lysates were incubated for 5 min at 95°C before running equal volumes (23 μl for the EV protein lysate and 45 μg for the cellular protein lysate) on a 12% polyacrylamide gel under non‐reducing conditions. Separated proteins were transferred onto PVDF membranes, which were then blocked in TBS‐T containing 2.5% BSA. Membranes were incubated with primary antibodies at 4°C overnight to detect CD9 and Calnexin. The following primary antibodies were used: mouse anti‐CD9 (1:1000, BioLegend, Cat. # 312102) and rabbit anti‐Calnexin (1:1000, Abcam, Cat. # ab75801). The following day, membranes were washed 3x with TBS‐T and incubated with horseradish peroxidase‐coupled secondary antibodies (1:5000, Cell Signalling, Cat. # 7074 + 7076) for 60 min at room temperature. After washing three times with TBS‐T and a short wash in PBS, the developer solution (SuperSignal West Dura, ThermoScientific, Cat. # 34076) was added. Labelled proteins were visualised by the Chemidoc Touch Imaging System (Biorad, Hercules, CA).