Nta nanosight lm10

Size distribution and concentration of EVs were analyzed by a NTA NanoSight LM10 (Malvern Instruments, Malvern, UK) analyzer, equipped with a 405 nm laser (Nano-Sight, Malvern Instruments, Malvern, UK). The suspension of EVs was diluted 10,000 times. Measurements were carried out at 25 °C, the samples were measured four times. Data analysis was performed using NTA 2.3 software.

Isolation of extracellular vesicles (EVs)was performed by differential ultracentrifugation, as described previously [25 (link)]. The pellet obtained by ultracentrifugation at 100,000 g for 70 min corresponding to the fraction containing the EVs was resuspended in 0.1 μm filtered PBS with sucrose at 1% (Sigma-Aldrich). The presence of EVs in the sample was detected by Transmission Electron Microscopy (TEM) JEM-1011 (JEOL, Japan) at 100 kV with a previous fixing with 2% paraformaldehyde and dyed with 2% phosphotungstic acid. The EVs and Taxol-encapsulated EVs were characterized by a Pierce™ bicinchoninic acid assay kit (Thermo Fisher Scientific, USA) for total protein quantification. The size distribution and particle concentration were determined by Nanoparticle Track Analysis (NTA) Nanosight LM10 (Malvern Panalytical, United Kingdom).
EVs were characterized by flow cytometry using specific antibodies: anti-CD9 antibody (FITC) (Abcam, United Kingdom), anti-CD63 (APC) (Bio-Rad, USA) and anti-CD81 antibody (PE) (Bio-Rad, USA). Also, the Molecular Probes™ CellTrace™ Calcein Violet, AM (Invitrogen, USA) was used to ensure EVs integrity. The fluorescence was monitored by a Cytoflex S Flow Cytometer using a Violet Laser (405 nm) and its light Side Scattering (SSCviolet).

The size and concentration of EVs were determined by NTA using the NTA NanoSight® LM10 (Malvern Instruments) analyzer, equipped with a blue laser (45 mW at 488 nm) and a C11440-5B camera (Hamamatsu Photonics K.K., Japan). Recording and data analysis were performed using the NTA software 2.3. The following parameters were evaluated during analysis of recording monitored for 60 s: the average hydrodynamic diameter, the mode of distribution, the standard deviation, and the concentration of vesicles in the suspension. Before NTA measuring, an aliquot of the isolated vesicles was thawed at room temperature and diluted with deionized water in 10, 100, 1000 times. The measurements were performed at least three times.