Nanoparticle tracking analysis (NTA) of EVs using the NanoSight NS300

EVs were analyzed by nanoparticle tracking analysis (NTA) using the NanoSight NS300 (Malvern Panalytical), as previously described by our group with slight modifications [6]. Twenty milliliters of EV-rich CCM was used. Following EV purification as described in the “EV purification from the EV-rich hBM-MSC-conditioned medium collected from the hollow-fiber bioreactor system” section, the EV pellet was suspended in 1 mL of filtered D-PBS^−/−. Hundred microliters of the 1-mL EV-rich sample was used for NTA and diluted 10–40× in filtered D-PBS^−/− to obtain a final volume of 1 mL of EV sample for analysis. Each sample was vortexed prior to filling the syringe with the sample and the syringe pump from Harvard Apparatus (cat.# 98-4730) was used for acquisition in flow mode. Each 1-mL sample was run using the following script: six captures of 1 min at speed 10 under flow mode. For capture settings, a camera level of 15 was used for all samples and a detection threshold of 30 was used for analysis resulting in approximately 30 particles per frame. Analysis of the raw data was performed using the NTA 3.0 software (Malvern Instruments) where analysis of five out of six captures was performed, removing the first capture to generate the approximate total EV concentration. Following NTA data analysis using the NTA 3.0 software, Excel was used to account for the dilution factor 10.