Anx5-positive EVs were enumerated by EM after sedimentation on EM grids as previously described [28 (link)]. Briefly, PFP samples were labeled with Anx5-conjugated gold nanoparticles (Anx5-gold-NPs) [45 ] as follows: 100 μL PFP were mixed with 1 μL 1 mm PPACK, 1 μL Anx5-gold-NPs at 1-3 × 1016 NP/L, supplemented with 10 mm Ca2+, and incubated for 30 min at ambient temperature. Samples were then diluted 30× with 100 mm cacodylate buffer, pH 7.4, containing 2 mm Ca2+, and deposited into 4.5 mL polyallomer centrifuge tubes containing at the bottom a 12-mm diameter hemispherical epoxy resin support onto which four EM grids coated with a continuous carbon film had been fixed (Fig. S1 ). Samples were centrifuged in an Optima™ MAX-E Ultracentrifuge equipped with a MLS50 rotor at 100 000 × g for 1 h at 20 °C, after which the liquid above EM grids was discarded, and EM grids were recovered and air dried.
Grids were observed with a CM120 microscope (FEI, France) operated at 120 kV. Images were recorded with a USC1000-SSCCD camera (Gatan, Warrandale, PA, USA).
A stringent method was applied for counting Anx5-positive EVs, in which only objects that presented a high and homogenous gold-NP labeling with a well-defined shape were taken into account.
Grids were observed with a CM120 microscope (FEI, France) operated at 120 kV. Images were recorded with a USC1000-SSCCD camera (Gatan, Warrandale, PA, USA).
A stringent method was applied for counting Anx5-positive EVs, in which only objects that presented a high and homogenous gold-NP labeling with a well-defined shape were taken into account.