Figure 4

Size exclusion chromatography removal of unbound dye increases the signal to noise ratio of CFSE-labeled EVs. (A) Representative plots of fluorescence detection on unstained EVs, CFSE-labeled EVs and EV-lacking controls, analyzed by nanoFACS. Note a shift on the background reference noise fluorescence, due to the presence of free fluorescent CFSE dye in the sample^{22, 23}. (B) Representative dot plots depicting 488-SSC and CFSE fluorescence showing unstained and CFSE-stained EVs before and after size exclusion chromatography. Dotted lines in A and B separate the CFSE^- and CFSE⁺ events, and were set on the limit of the background reference noise population in the unstained EV plot. (C) EV concentration analysis by NTA (in red) and total event rate by nanoFACS (in blue) of each fraction collected after size exclusion chromatography. NTA data shows the mean of three acquisitions and SD. (D) Ratio between EV and background reference noise median fluorescence intensity after eluting from size exclusion column (gated as in Fig. 2E). Dashed line shows the ratio before chromatography. Analysis of size exclusion chromatography fractions was performed twice with similar results. (E) ROC curves to assess specificity and sensitivity of EV identification by fluorescence. EV, extracellular vesicle; FSC, forward and SSC, side light scatter; MedFI, Median Fluorescence Intensity. SEC, size exclusion chromatography; ROC, Receiver Operating Characteristic; AUC, Area Under the Curve; FSC, forward scatter; SSC, side scatter.