Characterization of nanoparticles and fluorescent recombinant extracellular vesicles using three different generations of high-sensitivity flow cytometers.
5 affiliations
- 1Division of Cell Biology, Metabolism and Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3584 CM, The Netherlands.
- 2Division of Infection & Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3584 CL, The Netherlands.
- 3MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, China.
- 4Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent 9000, Belgium.
- 5Cancer Research Institute Ghent, Ghent 9000, Belgium.
Abstract
Aim: High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Methods: Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 113 and 155 nm) by using light-scatter-based detection thresholds [side scatter (SSC), forward scatter (FSC), violet side scatter (VSSC)] across a concentration range from 10⁶ to 10⁹ particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter-based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding. Results: Upon qualitative and quantitative analysis, we observed that instruments differed in sensitivity, the NF could detect 68 nm SiNPs, while both IF and CF were able to detect down to 91 nm SiNPs when using a scatter-based threshold, which was improved by using FSC and VSSC compared to SSC, respectively. We show that the NF required a higher sample concentration to ensure optimal detection, while IF and CF benefited from more diluted samples. Next, we defined a single particle detection range measuring fluorescent rEV and demonstrated that fluorescence-based detection improved the detection of particles of interest due to lower background interference. Conclusion: We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.
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BibTeX
@article{lozanoandres2026characterization,
title = {{Characterization of nanoparticles and fluorescent recombinant extracellular vesicles using three different generations of high-sensitivity flow cytometers.}},
author = {Estefanía Lozano-Andrés and Ye Tian and Sten F W M Libregts and An Hendrix and Xiaomei Yan and Ger J A Arkesteijn and Marca H M Wauben},
journal = {Extracellular vesicles and circulating nucleic acids},
year = {2026},
month = aug,
doi = {10.20517/evcna.2026.33},
url = {https://europepmc.org/article/MED/42898755},
note = {CC BY},
}
RIS
TY - JOUR
TI - Characterization of nanoparticles and fluorescent recombinant extracellular vesicles using three different generations of high-sensitivity flow cytometers.
AU - Estefanía Lozano-Andrés
AU - Ye Tian
AU - Sten F W M Libregts
AU - An Hendrix
AU - Xiaomei Yan
AU - Ger J A Arkesteijn
AU - Marca H M Wauben
PY - 2026
DA - 2026/08/18
JO - Extracellular vesicles and circulating nucleic acids
DO - 10.20517/evcna.2026.33
UR - https://europepmc.org/article/MED/42898755
AB - Aim: High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Methods: Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 113 and 155 nm) by using light-scatter-based detection thresholds [side scatter (SSC), forward scatter (FSC), violet side scatter (VSSC)] across a concentration range from 10⁶ to 10⁹ particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter-based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding. Results: Upon qualitative and quantitative analysis, we observed that instruments differed in sensitivity, the NF could detect 68 nm SiNPs, while both IF and CF were able to detect down to 91 nm SiNPs when using a scatter-based threshold, which was improved by using FSC and VSSC compared to SSC, respectively. We show that the NF required a higher sample concentration to ensure optimal detection, while IF and CF benefited from more diluted samples. Next, we defined a single particle detection range measuring fluorescent rEV and demonstrated that fluorescence-based detection improved the detection of particles of interest due to lower background interference. Conclusion: We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.
SN - 2767-6641
ER -