Characterization of nanoparticles and fluorescent recombinant extracellular vesicles using three different...

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.