Fluorescent Extracellular Vesicle Purification and Analysis

Freshly labelled fluorescent LEVs and SEVs from 1 x 10⁷ oocysts were diluted in 0.3 ml of PBS. Then, fluorescent samples were mixed with 1 ml of 60% iodixanol solution (OptiprepTM, Sigma-Aldrich), overlaid with 0.5 mL of 40%, 0.5 mL of 30% and 1.8 mL of 10% of iodixanol solutions and floated into the gradient by ultracentrifugation in a SW60Ti rotor (Beckman) at 192,000 × g, for 18 h, stopping without brake. After centrifugation, 12 fractions of 330 µl were collected from the top of the tube, diluted 40- to 80-fold with PBS and analysed by FC. Fraction densities were determined by refractometry. Gradient solutions were produced from the working solution (WS) by dilution with the HM solution. WS was prepared by mixing 5 vol of OptiPrep™ with 1 vol of 0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl, pH 7.4; HM solution also contained 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4. Gradient fractions of fluorescent LEVs and SEVs were analysed by FC with a Gallios Flow Cytometer (Beckman Coulter) using an optimised procedure, as previously described (Coscia et al., 2016 (link)).