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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Larger extracellular vesicles, microparticles (MPs) or microvesicles (MVs), especially their acquisition and characterization by flow cytometry (FACS), is increasingly in focus of clinical/translational research efforts. Several laboratories have shown that MPs/MVs might be suitable for the diagnosis and predicting prognosis in various diseases including cancer. However, FACS staining of larger extracellular vesicles (EVs) can be difficult and results potentially in false positive and inconsistent data interpretation. Despite that FACS equipment is well maintained and the operators have ample experience, a reliable and for larger EVs optimized staining protocol is missing. Here, we aim to close that gap and provide a working multi-antibody staining protocol for larger EVs isolated from human serum samples. We describe in detail the needed steps as currently done in our laboratory. Staining is demonstrated exemplarily for multi-antibody mix including CD147 , a potential cancer marker if applied in combination with other MP/MV surface markers.