Sc74566

An amount of 100 μL of plasma and 3 mL of urine samples were used for EV isolation using ExoGAG. The pellet containing EVs was resuspended in 200 μL of PBS and incubated for 1 h at 4 °C with antibodies against EVs’ protein markers [36 (link)]: CD9 (1:50, sc13118, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD63 (1:50, sc5275, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and flotillin-1 (1:50, sc74566, Santa Cruz Biotechnology, Santa Cruz, CA, USA). An isotype IgG antibody (1:50, 400120, BioLegend, San Diego, CA, USA) was used as negative control and added in equivalent volumes. Subsequently, an anti-mouse Alexa 488 (1:1000, ab150113, Abcam, Cambridge, UK) secondary antibody was added and stained for 1h at 4 °C in the dark. EVs were then washed once with PBS by centrifugation at 3000× g for 15 min at 4 °C and analyzed by flow cytometry (BD FACSAriaTM IIu, BD biosciences, San José, CA, USA).

5–20 μg of protein equivalents of extracellular vesicles (either untreated or previously treated with Lipofectamine) was placed on 18-mm coverslips with a poly-d-lysine coating (NeuVitro) and incubated with 400 μl of DMEM. After overnight incubation in 5% CO₂ and 37 °C, the medium was aspirated, and the coverslips were directly fixed with 1 ml of 2% paraformaldehyde in PBS for 15 min at room temperature and then washed with PBS. The coverslips were then blocked with 3% BSA in PBS, followed by standard immunofluorescence with the anti-exosome markers AIP1/Alix (1:200, ABC40, Millipore) and flotillin-1 (1:50, sc-74566, Santa Cruz Biotechnology), which were detected with Alexa Fluor 555 goat anti-mouse (1:500, A21424, Life Technologies) and Alexa Fluor 488 goat anti-rabbit antibodies (1:500, A11034, Life Technologies). Coverslips were mounted on Superfrost Plus slides (Menzel-Glaser) using Vectashield Antifade mounting medium (Vector). Fluorescence images were obtained at ×63 with a ×2–3 zoom using a Zeiss LSM 710 confocal microscope.