Zetasizer nano zs90 particle sizer

The size of the exosomes and microvesicles was determined by dynamic light scattering (DLS) using a Zetasizer nano ZS90 particle sizer (Malvern Instruments, Worcestershire, UK) at a 90° fixed angle. The particle diameter was calculated using the Stokes–Einstein equation. For particle sizing in solution, exosome and microvesicle aliquots were diluted in 10% PBS and analyzed at a constant 25 °C.

Exosome size was determined by DLS using a Zetasizer nano ZS90 particle sizer at a 90° fixed angle (Malvern Instruments, Worcestershire, UK). The particle diameter was calculated using the Stokes–Einstein equation. For particle sizing in solution, exosome aliquots were diluted in 10% PBS and analyzed at a constant 25 °C.

To collect intact exosomes, the exoRNeasy Serum/Plasma Midi kit protocol was used: in the last step of the process, QIAzol was substituted with 200 μL of Buffer XE, a reagent enabling elution and collection of exosomes. Exosome size was evaluated using the zetasizer nano ZS90 particle sizer (Malvern Instruments, Worchestershire, UK). Retrieved exosomes were then analyzed for the presence of CD9 and GD2 markers by flow cytometry after vesicles adsorption onto latex beads, as previously described [69 (link)]. Briefly, collected exosomes were incubated with 2 μL of 4 μm diameter aldehyde/sulfate latex beads (Invitrogen, Life Technologies Italia, Monza, Italy) for 2 hours at RT and then incubated for 30 min at RT in PBS supplemented with 2% FBS. Exosomes-coated beads were incubated for 30 min at 4 °C with primary mouse anti-human PE-conjugated monoclonal antibodies (mAbs) to CD9 or GD2. An isotype-matched PE-conjugated primary mAb was used as negative control. All mAbs were used in accordance with the manufacturer instructions. Samples were analyzed by Gallios flow cytometer and Kaluza software (Beckman Coulter, Milano, Italy).