Figure 5.
Imaging Flow Cytometry facilitates discrimination between single and coincident sEVs. (a) Serial dilution experiment showing measured linear concentration values of SSC(low)eGFP(+) sEVs for 2-fold dilutions without notable changes in measured mean fluorescence intensities (MFI) for this population. (b) Visualisation of single, fluorescent spots appearing differently in terms of brightness and size/pixel coverage within the SSC(low)eGFP(+) sEV population. (c) Application of different masking settings combined with the spot count feature on selected events showing sEV coincidence in order to evaluate their suitability for coincidence detection. Yellow digits indicate the number of events as calculated by the spot count feature depending on the masking setting applied. The intensity mask (intensity 14-4095) was the only one correctly masking all fluorescent spots in all images. (d, e) Plotted values using the spot count feature combined with the intensity mask on samples acquired at a concentration of 3.5 × 108 (d) and at 7.7 × 106 SSC(low)eGFP(+) events/mL (e), respectively. (f) Events gated on spots classified as single, double or triple based on spot count values from plots as shown in (d, e). (g) Enlarged image of event 22,943 demonstrating that also faint spots being very close to each other are counted correctly as distinct spots/sEVs. All measurements were performed with THP-1:CD63eGFP derived sEVs prepared as described in Figure 2.