Fig. 2

Live cell fluorescence imaging and trafficking of exosomes using dual-reporters. A Human 293T cells were transfected with Exo-gLuc-GFP and confocal images were recorded after 72 h of transfection. Images reveal that fluorescence signals (white arrows) were localized to cytosol, exhibiting punctate patterns. The nuclei of cells were stained blue by Hoechst reagent. B The colocalization of Exo-gLuc-RFP (red; white arrows) with exosome markers (green; white arrows: CD63-GFP, CD9-GFP, or CD81-GFP) in living human 293T cells was recorded after 72 h of transfection. The convergence of red and green fluorescence signals observed in the cytosol is demonstrated by yellow signals in the overly images (right column). C Confocal images of isolated exosomes from either non-modified control or Exo-gLuc-GFP transfected cells. D Gaussia luciferase activity was measured in PBS as well as in exosomes isolated from non-modified control and Exo-gLuc-GFP transfected cells. Data represents averages of two independent exosome preparations (n = 2). E Nanoparticle tracking analysis shows the vesicle size and distribution of exosomes isolated from either non-modified control and Exo-gLuc-GFP transfected cells. For each exosome sample, three video recordings were obtained using a NanoSight LM10 instrument equipped with a 405 nm, 60 mV laser source. NTA software was utilized to analyze and visualize exosome size and particle distributions. TLI: transmitted light image. Scale bar: 10 µm