Introduction: Extracellular vesicles (EVs) are small membrane-enclosed particles that play a key role in intercellular communication. Despite their increasing importance, their analysis remains challenging due to technical limitations in sample preparation. Interferometric light microscopy (ILM) represents a promising method for EV characterization but requires careful optimization of blood sample dilution and centrifugation procedures. Purpose: The main goal of the study was to determine optimal conditions for preparing blood samples for EV analysis using ILM, focusing particularly on the effects of different dilution levels and centripetal accelerations of the centrifuge rotor. We specifically wanted to test the hypothesis that centrifugation at accelerations up to 1000g (where g = 10 m/s2) does not significantly affect EV number density or hydrodynamic diameter. Methods: The study used blood samples from three donors. Samples were diluted with physiological saline at ratios of 2×, 5×, 10× and 20×, and centrifuged at five different accelerations (1g, 50g, 500g, 1000g and 10,000g). EV analysis in supernatants was performed using ILM, which enables measurement of particle density and hydrodynamic diameter. The molecular composition of supernatants was evaluated by spectrophotometry in ultraviolet and visual range (UV-vis) at wavelengths of 280 nm and 260 nm. Results: EV number density increased with centrifugal acceleration up to 500 g, where it reached maximum values, then decreased at higher accelerations due to EV sedimentation. The hydrodynamic diameter of EVs (average 120-130 nm) did not change significantly with different centripetal accelerations of the centrifuge rotor. At low dilution level (<10×), we observed apparent increases in hydrodynamic diameter, which we attribute to more concentrated solutions. The A280/A260 absorbance ratio, reflecting relative protein and nucleic acid content, did not change with different dilution levels or centripetal accelerations of the centrifuge rotor. Discussion and conclusion: The results confirmed our hypothesis that centrifugation at accelerations up to 1000g does not substantially affect EV properties. As optimal conditions for preparing blood samples for ILM-based EV analysis. We recommend centrifugation at 500g and starting with 10× dilution. These findings are important for standardizing methodology in clinical EV research, although the study's small sample size requires further validation with larger cohorts. Additionally, our findings show that potential artifacts in hydrodynamic diameter measurements must be considered when using low dilution levels.
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