Morphology of the isolated extracellular vesicles (EVs) was assessed by transmission electron microscopy (TEM) as described previously [26 (link)]. Specific markers for exosomes, CD9 or CD63 were detected as described earlier [26 (link)] using 10 μL of vesicles mixed with 10 μL of 0.5% bovine serum albumin in PBS and 3 μL (100 μg/mL) of corresponding monoclonal antibodies (Abcam, Cambridge, UK). All grids were examined in JEM 1400 (Jeol, Tokyo, Japan) TEM supplied with digital camera Veleta (EM SIS, Muenster, Germany). The measurements were made directly on the camera screen using iTEM (EM SIS, Muenster, Germany) software, version 5.2.
The size and concentration of the isolated EVs were determined by NTA using the NanoSight® LM10 (Malvern Instruments, Malvern, Worcestershire, UK) analyzer, equipped with a blue laser (45 mW at 488 nm) and a C11440-5B camera (Hamamatsu Photonics K.K., Shizuoka, Japan), at several dilutions according to the manufacturer’s instructions. Each sample was measured in triplicate, with a camera setting of 15, an acquisition time of 60 s, and a detection threshold setting of 5. At least 200 completed tracks were analyzed per video. NTA analytical software version 2.3 (Malvern Instruments, Malvern, Worcestershire, UK) was used for data analysis and capture.
The size and concentration of the isolated EVs were determined by NTA using the NanoSight® LM10 (Malvern Instruments, Malvern, Worcestershire, UK) analyzer, equipped with a blue laser (45 mW at 488 nm) and a C11440-5B camera (Hamamatsu Photonics K.K., Shizuoka, Japan), at several dilutions according to the manufacturer’s instructions. Each sample was measured in triplicate, with a camera setting of 15, an acquisition time of 60 s, and a detection threshold setting of 5. At least 200 completed tracks were analyzed per video. NTA analytical software version 2.3 (Malvern Instruments, Malvern, Worcestershire, UK) was used for data analysis and capture.