Female NMRI or C57BL/6 mice were used. Freshly purified DiR-labelled EVs were injected through the tail vein for intravenous (i.v.) injections for most experiments. Intraperitoneal (i.p.) and subcutaneous (s.c.) injections were carried out as well, where indicated. Biodistribution of DiR-labelled EVs was examined using 3 different doses: 1.5×1010 particles/gram body weight (p/g), 1.0×1010 p/g and 0.25×1010 p/g, the particle count was measured with NTA and the samples were diluted accordingly. For analysis of DiR-EVs distribution, IVIS Spectrum (Perkin Elmer) was used. IVIS spectrum is an instrument that contains a high-sensitive CCD camera, which enables both fluorescence and luminescence measurements. Here, either live (isoflurane sedated) mice were imaged or the animals were sacrificed and the organs harvested prior to analysis. For the perfusion experiment, the mice were sedated and the vascular system was flushed by transcardial perfusion for 5 minutes. The left ventricle was infused with PBS (5 ml/min) and the right atrium was perforated. The outflow liquid, liver and tail were monitored during the procedure to assure successful perfusion. After 5 minutes of perfusion, the organs were harvested and analysed. The live mice or the harvested organs were imaged for 1–2 seconds (excitation 710 nm, emission 760 nm). The data were analysed with the IVIS software (Living Image Software for IVIS®). For fluorescent micro computed tomography (µCT)-images, mice were secured in a mouse imaging shuttle, imaged in the IVIS for 3D fluorescence (FLIT) and transferred to the Quantum FX micro CT Imaging System (PerklinElmer, MA, USA) for CT-imaging. Co-registration of FLIT with the CT-scan was generated using Living Image software (30 (link),31 (link)). Adobe Photoshop CS4 and Adobe Illustrator were used to crop out and align the organ images.
Injection of CD63-EGFP EVs: CD63-EGFP EVs were generated, isolated and characterized by NanoSight as described above. Twenty four hours post-injections (2.9×1010 p/g), the organs were harvested and prepared as described below. The animal experiments were approved by the British and the Swedish local boards for laboratory animals.
Injection of CD63-EGFP EVs: CD63-EGFP EVs were generated, isolated and characterized by NanoSight as described above. Twenty four hours post-injections (2.9×1010 p/g), the organs were harvested and prepared as described below. The animal experiments were approved by the British and the Swedish local boards for laboratory animals.
