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. Author manuscript; available in PMC: 2021 Oct 27.
Published in final edited form as: ACS Nano. 2020 Sep 25;14(10):12732–12748. doi: 10.1021/acsnano.0c03064

Figure 1. Isolation and characterization of keratinocyte-derived exosomes from murine skin.

Figure 1.

(A) Quantification of extracellular vesicles and exosomes from murine skin and day 5 wound-edge tissue of C57BL/6 mice. (n=11, 12) (B) Schematic diagram of keratin 14 (K14) promoter-driven recombinant plasmids encoding CD9, CD63 or CD81 with “in frame” GFP reporter. (C) Representative scanning electron microscopic images of tissue nanotransfection (TNT) chip 2.0. (D) Schematic diagram showing the delivery of the three K14 promoter-driven plasmids via TNT in the dorsal murine skin. (E) Confocal microscopic images showing presence of keratinocyte derived exosome (Exoκ-GFP) in dermis (left) and super-resolution confocal microscopic images showing GFP labelled exosome (right). The white dash line shows epidermal-dermal junction. Scale, 10μm and 2μm. (F) Schematic diagram showing exosome isolation process from murine skin tissue post-TNT with the three plasmids. MP: membrane particles; AB: apoptotic bodies. (G) Particle size distribution of keratinocyte-derived exosomes (Exoκ−GFP), non-keratinocyte-derived exosomes (Exonon-GFP), and flow through (membrane particles and apoptotic bodies) from murine skin. (n=10) (H) Representative Scanning Electron Microscopy (SEM) images of murine keratinocyte-derived exosomes. Scale, 100 nm. (I-J) Binding of TSG-101 PE with the murine keratinocyte-derived exosome was further tested by autocorrelation curves as determined by fluorescence correlation spectroscopy (FCS) (I) and time-resolved fluorescence anisotropy (J). (n=10) (K) Flow cytometric analysis of murine skin keratinocyte-derived exosome on GFP-trap magnetic beads showing binding of TSG-101 PE, Alix-FITC, Flotillin 1-PE and HSP90-FITC antibodies. The histograms demonstrate the shift in fluorescence after binding of murine Exoκ-GFP on the GFP-trap magnetic beads with the respective antibody. All data were shown as mean ± SEM. Data in A were analyzed by two-tailed unpaired Student’s t-test. Data in J were analyzed by one-way ANOVA with the post-hoc Bonferroni’s multiple comparison test.