Figure 1. Quantitative exosome cell... | Rockefeller University Press

Figure 1.

$Figure 1. Quantitative exosome cell uptake dynamics. (a–c) HEK293 CD63-emGFP exosome uptake quantification using quantitative high content screening. Error bars: SD of three independent biological replicates. avg, average. (d) Human primary fibroblasts after incubation with 100 pM HEK293 CD63-GFP exosomes (green) for 10 min imaged by confocal fluorescence microscopy with DIC imaging. (e) CD63-emGFP HEK293 exosomes were copurified with CD63-mCherry exosomes and imaged by CLSM after spotting onto coverslips. The number of emGFP (G), mCherry (R), and GFP/mCherry (RG) double-positive vesicles was derived based on colocalization quantification. Vesicles were detected as light diffraction-limited emGFP or mCherry fluorescent spots of uniform size corresponding to the point spread function of the microscope with a negligible fraction of emGFP/mCherry double-positive spots, confirming recovery of single vesicles (left). Exosomes from CD63-emGFP and CD63-mCherry double-transfected cells yielded ∼40% of double-labeled vesicles (middle and right). Data representative of three independent experiments. (f) High-resolution 3D images of Huh7 cells incubated with 50 pM CD63-emGFP exosomes for 2 h recorded by live cell confocal microscopy (red: CellMask DeepRed). (g) Quantification of exosomes localizing to the cell membrane versus the cell interior (Error bars: SD of three independent biological replicates, five cells per field of view; p-values, analysis of variance: ***, P < 0.001; ****, P < 0.0001). (h) SPT of CD63-emGFP/CD63-mCherry HEK293 exosome uptake in primary human fibroblasts (confocal live cell imaging, 50 s/z-stack). Trajectory statistics in Fig. S1 e.$

Quantitative exosome cell uptake dynamics. (a–c) HEK293 CD63-emGFP exosome uptake quantification using quantitative high content screening. Error bars: SD of three independent biological replicates. avg, average. (d) Human primary fibroblasts after incubation with 100 pM HEK293 CD63-GFP exosomes (green) for 10 min imaged by confocal fluorescence microscopy with DIC imaging. (e) CD63-emGFP HEK293 exosomes were copurified with CD63-mCherry exosomes and imaged by CLSM after spotting onto coverslips. The number of emGFP (G), mCherry (R), and GFP/mCherry (RG) double-positive vesicles was derived based on colocalization quantification. Vesicles were detected as light diffraction-limited emGFP or mCherry fluorescent spots of uniform size corresponding to the point spread function of the microscope with a negligible fraction of emGFP/mCherry double-positive spots, confirming recovery of single vesicles (left). Exosomes from CD63-emGFP and CD63-mCherry double-transfected cells yielded ∼40% of double-labeled vesicles (middle and right). Data representative of three independent experiments. (f) High-resolution 3D images of Huh7 cells incubated with 50 pM CD63-emGFP exosomes for 2 h recorded by live cell confocal microscopy (red: CellMask DeepRed). (g) Quantification of exosomes localizing to the cell membrane versus the cell interior (Error bars: SD of three independent biological replicates, five cells per field of view; p-values, analysis of variance: ***, P < 0.001; ****, P < 0.0001). (h) SPT of CD63-emGFP/CD63-mCherry HEK293 exosome uptake in primary human fibroblasts (confocal live cell imaging, 50 s/z-stack). Trajectory statistics in Fig. S1 e.