Exosomes are taken up at the filopodia base and shuttle with endosomes. (a) 399 individual exosome cell entry events in human primary fibroblasts documented by DIC/CLSM or TIRF microscopy were classified as indicated (pooled data from 13 independent experiments, including Videos 4, 5, and 6). (b) Ernie and Bert illustrate typical examples for exosomes classified as either filopodia surfing or entering at the filopodia base, respectively. (c) Filopodia perturbation of human primary fibroblasts pretreated for 60 min with 40 µM SMIFH2 or DMSO at 0.5%, monitored by DIC imaging over 90 min of recovery, and (d) cumulative representation of exosome uptake trajectories in SMIFH2 versus DMSO pretreated fibroblasts by CLSM/DIC live cell imaging (exosomes at 30 pM). (e) Quantification of exosome uptake in SMIFH2 or DMSO pretreated fibroblasts by automated high content screening (exosomes at 30 pM, 2 h), as compared with exosome uptake inhibition by addition of Heparin (0.4%). Error bars: SD from three biological replicates; p-values: analysis of variance. avg, average; ***, P < 0.001; ****, P < 0.0001; ns, not significant. Representative examples of exosomes entering together with CellMask DeepRed into plasma membrane-derived endocytic vesicles (f and g) which can be visualized by DIC (h). All Images represent single frames from DIC/CLSM live cell movies. Red, plasma membrane labeling (CellMask Deep Red); green, CD63-emGFP HEK293 exosomes. Exosome trajectories color coded for speed. (i) TEM visualization of CD63-Apex2–tagged exosomes (30 pM) internalized into human primary fibroblasts (4 h) using DAB staining.