Extracellular Vesicle Isolation from Follicular Fluid

Extracellular vesicles isolated from FF and AOF were obtained via ODG UC [25 (link)]. Briefly, appropriate amounts of homogenization buffer (10-mM Tris-HCl, 1-mM EDTA, and 0.25-M sucrose (pH 7.4)) and iodixanol working solution were mixed to prepare gradients of 5%, 10%, 20%, and 40% iodixanol solutions. The iodixanol working solution was made by adding a solution buffer (60-mM Tris-HCl, 6-mM EDTA, and 0.25-M sucrose (pH 7.4)) to a stock solution of OptiPrep™ (60% (w/v) aqueous iodixanol solution). The gradient was prepared in a 16.8-mL open-top polyallomer tube (Beckman Coulter, Brea, CA, USA) by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10%, and 3.5 mL of 5% solutions on top of each other. The FF or AOF were overlaid onto the top of the gradient. Subsequently, the gradient was centrifuged at 4 °C for 18 h at 100,000× g (SW 32.1 Ti rotor, Beckman Coulter, Brea, CA, USA). Out of 16 layers of gradient fractions, EVs were mostly found in fractions 8 and 9 [25 (link)]; both fractions were pooled and diluted in 14-mL PBS and, subsequently, centrifuged for 3 h at 100,000× g and 4 °C. The resulting pellet was resuspended in 100-µL PBS and stored at −80 °C for further quantification and identification.