Sc 5275

Vesicles were characterized in terms of physical properties (size, concentration) by Nanoparticle tracking analysis (NTA) using the NanoSight NS300 (Malvern Instruments).
An additional flow cytometry analysis (BD FACSAria^TM IIu, BDbiosciences, SanJosé, CA, USA) was performed and tetraspanins CD63, and CD81 protein levels were analysed as the most usually employed markers for EV characterization [19 (link),24 (link)]; their presence demonstrates the lipid-bilayer structure specific of EVs. Semen EVs isolated by UC, ExoGAG or miRCURY cell/urine/CSF were incubated for 1 h with antiCD81 (1:50, sc7637, Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiCD63 (1:50, sc5275, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antiIgG (1:50, 400120, BioLegend) as negative control, followed by anti-mouse Alexa488 (1:1000, ab150113, Abcam, Cambridge, UK) as the secondary antibody as described [22 (link)]. Quantification of fluorescence was calculated as the fold-change of fluorescence intensity median with respect to IgG (immunoglobulin G) negative control. Positive signals were considered as those with a fold-change fluorescence intensity of two or higher than two.

Exosome isolation, characterization (by TEM and western blotting) and storage were performed as previously reported by us and others [27 (link),29 (link)]. In brief, starting from passage three, the MSCs culture medium was removed when the cells reached 80% confluency, and the serum-free medium was replenished and left for 24 h; it was collected and now called conditioned medium (CM). The collected CM was filtered through a 0.22 μm filter (to remove debris and dead cells) and was centrifuged at 10,000 g for 30 min at 4 °C and the supernatant was collected. The supernatant was then subjected to ultracentrifugation at 100,000 g for 60 min at 4 °C to pellet the exosomes. The pellet was washed twice with ice-cold PBS, and ultracentrifugation was performed for each wash to re-pellet the isolated exosomes. To quantify the amount of isolated exosomes, the protein content was measured by standard Bradford assay, and then the isolated exosomes were adjusted to 100 µg protein in 200 µL aliquots and stored at −80 °C. The characterization of isolated exosomes was performed using the detection of exosomes’ markers, CD63 (sc-5275, Santa Cruz Biotechnology, USA) and CD81 (sc-166029, Santa Cruz Biotechnology, Dallas, Texas, USA), by western blotting and by transmission electron microscopic (TEM) detection of their shape and size.

A mixture of Pierce Cell Lysis Buffer (Thermo Fisher, USA) and Halt Protease Inhibitor Cocktail (Thermo Fisher, USA) was used to lyse exosome samples. The samples were then processed by electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, USA). Mouse anti-CD63 (sc-5275, 1 μg/mL, Santa Cruz, USA), mouse anti-HSP90 (ab13492, 1 μg/mL, Abcam, USA), and rabbit anti-TSG101 (ab30871, 1 μg/mL, Abcam, USA) were used as primary antibodies for incubating the membrane at 4 °C overnight. Goat anti-mouse IgG (ab97040, 0.05 μg/mL, Abcam, USA) and goat anti-rabbit IgG (ab97080, 0.05 μg/mL, Abcam, USA) were used as secondary antibodies. Protein abundances were analyzed with a ChemiDoc XRS+ system (Bio-Rad, USA).

Equal amounts of proteins were separated by SDS-PAGE electrophoresis, transferred onto nitrocellulose membranes which were blocked with 5% milk in TBS with 0.1% Tween-20 for 1 h at room temperature. The membranes were incubated over night at 4°C with primary antibodies against CD63 (1 : 200, sc-5275 Santa Cruz), eukaryotic elongation factor 2 (eEF2) (1 : 1 000, 2332, Cell Signalling), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1 : 1000, 5174S, Cell Signalling), Hexokinase I (1 : 1000, C35C4, Cell Signalling), Hexokinase II (1 : 100, C64G5, Cell Signalling), MYCN (1 : 200, sc-53993, Santa Cruz), phosphofructokinase PFKP (1 : 1 000, 8164S, Cell Signalling), PKM1/2 (1 : 1 000, 3190S, Cell Signalling), PKM2 (1 : 1 000, 4053S, Cell Signalling), rib. L10a (1 : 500, sc-100827, Santa Cruz) and β-actin (1 : 1000, 3700, Cell Signalling). After washing with TBS 0.1% Tween-20 three times for 5 min at room temperature, the membranes were incubated with horseradish peroxidase (HRP)—conjugated secondary antibodies for 1 h at room temperature (anti-mouse-HRP, 1 : 10 000, sc-2031, Santa Cruz, anti-rabbit-HRP, 1 : 10 000, sc-2313, Santa Cruz). The membranes were washed three times for 5 min at room temperature with TBS 0.1% Tween-20 and incubated with enhanced chemiluminescent substrate (32106, Pierce). The protein bands were visualized with X-ray films.