Characterization of nanoparticles and fluorescent recombinant extracellular vesicles with three high-sensitivity flow cytometers

Abstract

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, namely the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 114 and 155 nm) by using light-scatter based detection thresholds (SSC, FSC, 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 for their qualitative and quantitative analysis. We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.