Gold particles

Fixed EV specimens (4% PFA in PBS mixed 1:1 with EV) were placed onto 10 min UV irradiated 300 mesh formvar/carbon coated grids and allowed to absorb to the formvar for 5 min. For immunogold staining, the grids were placed into 20 µL 0.01% Tween/PBS (10 min) and after that into a blocking buffer (0.5% fish gelatin with 0.1% ovalbumin in PBS) for a block step for 1 h. Without rinsing, the grids were immediately placed into the primary antibody (diluted in a blocking buffer) at the appropriate dilution overnight at 4 °C (1:100 anti-CD9 Abcam (Cambridge, UK), ab236630). As controls, some of the grids were not exposed to the primary antibody. The next day, all the grids were rinsed with PBS then floated on drops of the appropriate secondary antibody attached with 10 nm gold particles (AURION 1:30) for 2 h at room temperature (RT). Grids were rinsed 3 times with PBS and were placed in 1% glutaraldehyde (in PBS) for 5 min. After rinsing in PBS and distilled water, the grids were stained for contrast using 2% uranyl oxalate solution (pH 7 for 5 min in the dark). Afterwards, the grids were incubated in drops of methyl cellulose-uranyl oxalate (8 parts 2% methyl cellulose, 1 part ddH2O, 1 part 4% uranyl acetate (in water), pH 4, sterile filter) for 10 min on ice (dark) [61 (link)]. After that, the grids were removed with stainless steel loops and excess fluid was blotted by gently pushing on Whatman filter paper. After air-drying, the samples were examined and photographed with a Zeiss EM10 electron microscope (Zeiss, Jena, Germany) and a Gatan SC1000 Orius™ CCD camera (GATAN, Munich, Germany) in combination with the DigitalMicrograph™ software 3.1 (GATAN, Pleasanton, CA, USA). Images were adjusted for contrast and brightness using Adobe Photoshop CC 2018 (Adobe Systems, San José, CA, USA).

Platinum nanoparticles (Pt-PEG-17, referred to as Pt-NPs) were prepared as explained in the recently submitted French patent (FR 1900008). Briefly: Pt-NPs were synthetized by γ-ray water radiolysis of Pt containing salt and embedded with polyethylene glycol (PEG) to increase their biocompatibility. Pt-NPs were mainly spherical with an average platinum core diameter of 2.6 nm. Preliminary results were obtained also for gold nanoparticles (Au-NPs) which are composed of a Au core of 2.4 nm encapsulated by the dithiolated polyaminocarboxylate (DTDTPA) shell. For SkBr3 Au-NPs incorporation, 8 µL of 10 nm-sized gold particles (Aurion, Wageningen, The Netherlands) were added to the medium in each well 16 h prior to irradiation in order to obtain a maximum uptake in the cell cytoplasm via diffusion (Figure 6) [53 (link),75 (link)]. In other experiments 2.6 nm Pt-NPs or 2.4 nm Au-NPs were added to the medium 6 h before irradiation at 0.5 mM concentration.