Preparation of sEVs derived from PC3 cells and determination of their size and concentration. (A) sEVs from PC3 cells were isolated from the cell culture supernatant by ultrafiltration and ultracentrifugation. Tetraspanins tagged with Halo7 in sEVs were fluorescently labeled with SaraFluor650T (SF650T). (B) Negative-staining TEM images of sEVs revealed that the mean size of the sEVs was 83 ± 19 nm (mean ± SD). (C) The mean size of the sEVs determined by qNano was 69 ± 17 nm, as indicated by the arrowhead. (D) Single-particle fluorescence images of sEVs by TIRFM. Only when sEVs expressed CD63-Halo7, single particles labeled with SF650T (sEVs–CD63Halo7-SF650T) could be observed. (E, G, and I) We directly measured the number of sEV-tetraspanin-Halo7-SF650T particles bound to the antibody-coated glass. After incubating the sEV solution (2 × 10¹⁰ particles/ml) on antibody-coated glass at a dilution factor (df) of 1, 3, or 9, followed by three washes with HBSS, we obtained TIRFM images of single sEV–CD63-Halo7-SF650T particles. Single-particle fluorescence images of three concentrations of sEV–CD63Halo7-SF650T particles (E), sEV–CD81Halo7-SF650T particles (G), and sEV–CD9Halo7-SF650T particles (I), which attached to glass coated with anti-CD63 antibody, anti-CD81 antibody, and anti-CD9 antibody, respectively. df indicates the dilution factor. (F, H, and J) The number of sEVs bound to the glass decreased in accordance with the dilution factor (df), which allowed us to obtain a calibration curve. The numbers of sEV–CD63Halo7-SF650T particles (F), sEV–CD81Halo7-SF650T particles (H), and sEV–CD9Halo7-SF650T particles (J) at three df values attached to the CD63 antibody, anti-CD81 antibody, and anti-CD9 antibody-coated glass, respectively (n = 16 images). Data are presented as the mean ± SE. The sEV concentration was adjusted according to the calibration line.