2.4.1. Purification of Small Extracellular Vesicles by Differential Ultracentrifugation

Small EVs/exosomes were purified through differential centrifugation as previously described [14,26]. Briefly, serum samples were diluted with equal volumes of phosphate-buffered saline (PBS) to reduce fluid viscosity. Diluted samples were centrifuged at 2000× *g* at 4 ◦C for 30 min and pellets were discarded to remove cell contaminants. Subsequently, supernatants were centrifuged at 12,000× *g* at 4 ◦C for 45 min to remove apoptotic bodies, mitochondrial fragments, cell debris, and large vesicles (mean size > 200 nm). Supernatants were collected and ultracentrifuged at 110,000× *g* at 4 ◦C for 2 h. Pellets were recovered and resuspended in PBS, filtered through a 0.22-μm filter, and ultracentrifuged at 110,000× *g* at 4 ◦C for 70 min to eliminate contaminant proteins. Pellets enriched in purified sEVs were finally resuspended in 100 μL of PBS. To quantify sEVs, total protein concentration was measured using the Bradford assay [27].

#### 2.4.2. Western Immunoblot Analysis of Small Extracellular Vesicles

Western immunoblot analysis was performed to assess the purity of sEV isolation, to determine the type of sEVs on the basis of the expressed tetraspanins, and to characterize their protein cargo as previously described [14,28]. Briefly, equal amounts (1.25 μg) of sEV proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electroblotted onto polyvinylidenefluoride (PVDF) Immobilon-P (Millipore, Burlington, MA, USA). Membranes were probed with primary antibodies against tetraspanins CD63 (1:200), CD9 (1:200), CD81 (1:200), a specific cocktail of antibodies (1:250) targeting mitochondrial markers (Table 1), flotilin (1:200), and heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1; 1:1000). Technical specifications of primary antibodies used for Western immunoblotting are detailed in Supplementary Table S1.


**Table 1.** Mitochondrial components and related electron transport chain complexes assayed in purified small extracellular vesicles by Western immunoblotting.

Abbreviations: ATP5A, adenosine triphosphate 5A; ETC, electron transport chain; MTCOI, mitochondrial cytochrome C oxidase subunit I; NDUFB8, nicotinamide adenine dinucleotide reduced form (NADH):ubiquinone oxidoreductase subunit B8; NDUFS3, NADH:ubiquinone oxidoreductase subunit S3; SDHB, succinate dehydrogenase complex iron sulfur subunit B; UQCRC2, ubiquinol-cytochrome C reductase core protein 2.

The following day, membranes were incubated for 1 h at room temperature with anti-mouse peroxidase-conjugated secondary antibodies (1:2000) (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Blots were visualized using the Clarity Max ECL Western Blotting Substrate (Bio-Rad Laboratories) and images were acquired by the ChemiDoc MP Imaging System and analyzed by Image Lab TM software version 6.0.1 (Bio-Rad Laboratories). Values of optical density (OD) units of each protein band immunodetected were normalized for the amount of sEV total proteins, as determined by the Bradford assay, and related to the control group, whose OD was set at 100%.
