Characterization of nanoparticles and fluorescent recombinant extracellular vesicles using three different generations of high-sensitivity flow cytometers

Reliable characterization of extracellular vesicles (EVs) and nanoparticles (NPs) at the single-particle level remains challenging because their small size and weak light-scattering signals approach the detection limits of flow cytometers. Here, we performed a controlled cross-platform comparison of three flow cytometers representing different generations: NanoFCM, BD Influx, and CytoFLEX LX.

Using silica nanoparticles (SiNPs) with defined sizes of 68, 91, 113, and 155 nm, we evaluated different light-scatter detection thresholds and demonstrated substantial differences in sensitivity between instruments. NanoFCM detected 68 nm SiNPs, whereas BD Influx and CytoFLEX LX detected particles down to 91 nm using optimized scatter parameters. We further showed that sample concentration strongly affects particle discrimination and that optimal dilution differs between platforms.

Using fluorescent recombinant EVs, we demonstrated that fluorescence-based detection can improve the identification of particles of interest by reducing background interference and enabling more reliable particle quantification.

This work provides practical guidance for selecting detection strategies and sample concentrations across different flow cytometry platforms and establishes an important framework for the standardized characterization of nanoparticles and EVs.

The work entitled “Characterization of nanoparticles and fluorescent recombinant extracellular vesicles using three different generations of high-sensitivity flow cytometers” was published in Extracellular Vesicles and Circulating Nucleic Acids (published on August 18, 2026).