Characteristics of human semen exosomes. (A) Fluorescence-activated cell sorting (FACS) analysis of pooled (n =20) SE incubated with α-MHC-II antibody coated latex beads (4 μm) and stained for CD63 (Left, red histogram) and CD81 (Right, red histogram). Isotype control antibody is represented by black histograms. (B) Left, transmission electron microscopy (TEM) of pooled (n =20) semen exosome (SE) stained for CD63 (α-CD63) and detected by 6 nm gold-labeled secondary antibody (arrow). Right, TEM of pooled (n =20) SE stained for Calnexin (α- Calnexin) and counterstained with 10 nm gold-labeled secondary antibody. Calnexin was not detected. (C) Western blot of SE (n =5) and paired spermatozoa protein extracts, stained for CD63, Calnexin, and GAPDH. Pooled SE population (n =89) were categorized based on (D) size and (E) electron density. (F) SE protein concentrations from different donors range between 5.3 - 6.8 mg/ml of semen.