Refining Flow Cytometry-based Sorting of Plasma-derived Extracellular Vesicles

• Published in: Biological procedures online Vol. 27; no. 1; pp. 33 - 16
• Main Authors: Reverberi, Daniele, Ciferri, Maria Chiara, Rosenwasser, Nicole, Catino, Alessandro, Poppa, Giuseppina, Giusti, Ilaria, Dolo, Vincenza, Quarto, Rodolfo, Santamaria, Sara, Colombo, Monica, Coco, Simona, Tasso, Roberta
• Format: Journal Article
• Language: English
• Published: London BioMed Central 20.08.2025; BioMed Central Ltd; BMC
• Subjects: Biological Techniques; Biomarkers; Biomedical and Life Sciences; Biomedicine; Calibration; Cancer; CD9 antigen; Chromatography; Cloning; Ethylenediaminetetraacetic acid; Extracellular Vesicles; Flow Cytometry; Health aspects; Lasers; Metastases; Methodology; Plasma; Sorting; Sucrose; Surface markers; Tumor Biomarkers; Ultracentrifugation; Flow Cytometry; Tumor Biomarkers; Extracellular Vesicles; Sorting; Cancer
• ISSN: 1480-9222; 1480-9222
• DOI: 10.1186/s12575-025-00293-2

Background Extracellular vesicles (EVs) are membrane-bound particles crucial for intercellular communication and serve as promising biomarkers for diseases, including cancer. Isolating and characterizing specific EV subpopulations, particularly those in plasma/serum, enhances biomarker precision and supports targeted therapies. Cancer-derived EVs often express unique surface markers, enabling distinction from other EVs. Accurate sorting of tumor-associated EVs provides insights into cancer progression, metastasis, and treatment response. Results This study presents a robust method for isolating and sorting CD9 + plasma EVs as a proof-of-concept for broader EV subpopulation analyses. Plasma EVs were isolated via sucrose cushion ultracentrifugation, optimizing purity and yield. Flow cytometry with fluorescence threshold triggering was fine-tuned to detect and sort CD9 + EVs, with instrument calibration and parameter adjustments mitigating swarming and improving sorting accuracy. Size exclusion chromatography further enhanced efficiency by reducing background noise. Sorted CD9 + EVs retained size and marker expression, including Syntenin, Alix, Flotillin-1, and CD9, which were enriched post-sorting. Conclusions These advancements enable high-purity EV subpopulation isolation, facilitating applications such as identifying cancer biomarkers and developing EV-based targeted therapies.