Zetaview nanoparticle tracking analyzer nta

EVs sizes were measured using ZetaView nanoparticle tracking analyzer (NTA; Particle Metrix GmbH). For measurements, samples were diluted to 100, 500, 1000, and 2000 in previously filtrated PBS (0.22μm) for optimal concentration range for the NTA software (ZetaView Software version 8.02.31, Particle Metrix GmbH). Software parameters were the temperature at 23°C, the sensitivity of 30–85 frames per second (fps), a shutter speed of 55, and laser pulse duration equal to that of shutter duration. Acquisition parameters were set to a minimum brightness of 20, a maximum size of 200 pixels, and a minimum size of 5 pixels. Polystyrene particles (Microtrac GmbH) with an average size of 100 nm were used to calibrate the instrument before sample readings. Data were analyzed using ZetaView Software and Microsoft Excel 2013 (Microsoft Corp).

For characterization of the purified sEVs by using transmission electron microscopy (TEM), purified sEVs were dropped on a copper grid for 5 min. Then the copper grids were washed with PBS 3 times, followed by incubating with 1% v/v uranyl acetate for 40 s. Then copper grids were observed on a HT7700 TEM (Hitachi). The diameter and particle number of purified sEVs were determined by ZetaView nanoparticle tracking analyzer (NTA, Particle Metrix).