Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis

Research on extracellular vesicles (EVs) has intensified over the past decade, including fluorescent membrane labeling of EVs. An optimal fluorescent method requires the size of EVs to be preserved after labeling. Lipophilic fluorescent dyes, such as CellMask (TM) Green (CMG), have been widely used for this purpose. Here, we investigated conditions affecting the optimum CMG labeling of EVs derived from human choriocarcinoma cells (JAr) and different biological fluids using fluorescence NTA (fl-NTA). The effect of CMG labeling on the size, concentration and zeta potential (ZP) on JAr EVs purified with different methods were measured along with biological fluid-derived EVs. With the increase of CMG dye concentration, a significant decrease in the mean size of fluorescent nanoparticles (fl-NPs) was observed. The ZP of fl-NPs originating from JAr cells with the lowest and highest dye concentrations showed a significant shift towards more and less negative ZP values, respectively. Differences in the concentration of fl-NPs were observed for JAr EVs purified using size-exclusion chromatography (SEC) alone and SEC in combination with tangential flow filtration. The proportion of CMG labeling of NPs varied across different biological sources. CMG labeling may be a reliable technique for the detection of EVs using fl-NTA.

Automated summary

Research has shown that the concentration of CMG fluorescent dye affects the size of fluorescent nanoparticles (fl-NPs), with an increase in dye concentration resulting in a significant decrease in size. Zeta potential (ZP) values of fl-NPs from JAr cells also shift significantly towards more negative or less negative values with varying dye concentrations. Furthermore, the proportion of CMG labeling of NPs varies across EVs purified using different methods and from different biological sources.