Dedicated nanoparticle flow cytometry for single extracellular vesicle phenotyping: Performance of the CytoFLEX Nano

Gudbergsson, J. M.; Malle, M. G.; Jensen, J. B.; Truelsen, E. N.; Hemmingsen, J. K.; Nelson, S.; Kjems, J.; Hansen, A. S.; Etzerodt, A.; Bohn, A. B.

doi:10.64898/2025.12.15.694348

bioRxiv · 10.64898/2025.12.15.694348

Dedicated nanoparticle flow cytometry for single extracellular vesicle phenotyping: Performance of the CytoFLEX Nano

Gudbergsson, J. M.·Malle, M. G.·Jensen, J. B.·Truelsen, E. N.·Hemmingsen, J. K.·Nelson, S.·Kjems, J.·Hansen, A. S.·Etzerodt, A.·Bohn, A. B.

View original source ↗Export metadata ↓

Abstract

Accurate discrimination of extracellular vesicles (EVs) from non-vesicular nanoparticles and robust phenotyping of individual EVs directly within complex biofluids are essential to advance understanding of EV biology and to realize their potential as biomarkers and therapeutic agents. Conventional flow cytometers, originally designed for cellular analysis, lack the scatter and fluorescence sensitivity, dynamic range, and event-rate control required for quantitative characterization of nanoscale vesicles and are particularly susceptible to coincident (swarm) detection. Dedicated nanoparticle flow cytometers have been developed to address these limitations, and here we systematically evaluate the suitability of the CytoFLEX Nano (Beckman Coulter) for EV analysis. Using a series of calibration beads of different materials, fluorescent liposomes, and EVs isolated from peritoneal fluid and cell culture medium, we assess size-detection thresholds, scatter and fluorescence sensitivity, dynamic range, and volumetric counting accuracy. These data provide practical guidance for implementing nanoscale flow cytometry in EV research and support the informed adoption of dedicated nanoparticle cytometers in studies adhering to current EV reporting standards.

Explore related subjects

cell biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Gudbergsson, J. M., Malle, M. G., Jensen, J. B., Truelsen, E. N., Hemmingsen, J. K., Nelson, S., Kjems, J., Hansen, A. S., Etzerodt, A., Bohn, A. B.. 2025-12-17. Dedicated nanoparticle flow cytometry for single extracellular vesicle phenotyping: Performance of the CytoFLEX Nano. https://doi.org/10.64898/2025.12.15.694348

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Sequence context and partner availability specify divergent cell fates from a single transcription factor

Transcription factors drive the gene regulatory networks that control developmental transitions, yet which of their many targets a cell uses remains unresolved. Expressing Cebp, Cebp{beta} or Cebp{delta} identically, alone or with Oct4, Sox2, Klf4 and cMyc, gave four outcomes: adipocyte from fibroblasts and neural stem cells, trophectoderm from embryonic stem cells, and myeloid from B cells. Raising Cebp levels changed how many sites it reached, not which sequence it read: clustered motifs maintained somatic enhancers, while spillover onto low-copy sites created de novo enhancers carrying composites of Cebp and cell-type-restricted partners. In fibroblasts, Cebp also redistributed Oct4, Sox2 and Klf4 from early targets, blocking iPSC formation. Supplying the predicted partners rewrote the output: Spi1 redirected Cebp into parallel adipogenic and myeloid programs, via a transient PpargCD45 state using separate enhancers for each; TFAP2C abolished adipogenic conversion. Motif density and partner composites therefore assemble a factor's instructive output in the receiving cell.

cell biology↗

bioRxiv2026

Tracking the conformational dynamics of single molecules in live cells; GTPase cycling in Cdc42 gradients and microclusters

The Rho family of GTPases play a critical role in many cellular processes by acting as molecular switches relying on conformational changes to transition between active and inactive states. The spatial distribution and kinetics of these conformational changes are complex and vary across the cell. We describe here biosensors based on environment-responsive fluorescent dyes, capable of reporting the conformational changes of individual GTPase molecules as they traffic within living cells (SPARC Cdc42 and SPARC Rac1, 'Single Particle Activation Reporter Constructs'). We use SPARC Cdc42 to determine the rates of different steps in GTPase regulation, activation/inactivation and membrane insertion, that together generate localized gradients of Cdc42 activity driving cell motility. The biosensors also revealed heterogeneously distributed clusters of Cdc42, the mechanisms of their activation, and potential roles in polarized motility.

cell biology↗

bioRxiv2026

High-content screening identifies FDA-approved activators of mitophagy

Mitochondrial diseases are highly heterogeneous disorders caused by defects in oxidative phosphorylation, for which only limited therapeutic options are available. The elimination of dysfunctional mitochondria via mitophagy activation is an attractive strategy to treat several mitochondrial diseases, irrespective of the underlying genetic defect. To identify pharmacological activators of this pathway, we performed a high-content screening of 1,971 FDA-approved compounds using cellular reporters of TFEB nuclear translocation and mitophagy. This screening identified 294 compounds that promoted TFEB translocation, nine of which increased the number of mitolysosomes without compromising cell viability or damaging mitochondria. Among the selected compounds, Meptazinol and Ponatinib emerged as the top-ranked mitophagy inducers in fibroblasts from patients with complex I and IV deficiency, respectively, and were prioritised for further characterisation. However, they did not rescue the bioenergetic defect, suggesting that the stimulation of mitophagy per se is not sufficient to ameliorate the OXPHOS defect in mitochondrial diseases. Overall, our findings establish a scalable platform for the identification of pharmacological activators of TFEB and identify candidate compounds for therapeutic modulation of mitophagy.

cell biology↗