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. 2018 Jun 25;18:54–64. doi: 10.1016/j.redox.2018.06.009

Fig. 3.

Fig. 3

MDRC-derived exosomes contain polarized mitochondria. (a) Characterization of MDRC-derived exosomes after purification. Quantitation of MDRC-derived EVs that were negative for non-exosomal markers ARF6 and GRP94. Quantitation of percent MDRC-derived exosomes with Tetraspanins (CD63, CD9, CD81) and Tsg101, an ESCRT pathway molecule (no statistical significance observed; Student's T-test) (n = 3 healthy, n = 3 asthmatics; 3 technical replicates each) (b) Schematic for characterization and validation of Mito-GFP+exosomes derived from MDRCs. MDRC mitochondria were marked with MitoT-Green or Mito-GFP. MDRC-derived exosomes marked with Mito-GFP were labeled with MitoT-Red (c) ImageStream flow cytometry images of MDRC-derived exosomes positive for MitoT-Green, CD63, and HLA-DR (n = 3 healthy, n = 3 asthmatics; 3 technical replicates each) (d) Characterization of MDRC-derived Mito-GFP+ exosomes. BAL MDRCs were isolated by FACS sorting from n = 3 healthy and n = 3 asthmatic subjects. Purified MDRCs were transduced with Mito-GFP and exosomes isolated from cell culture supernatant at 48 h. Histogram showing quantitation of percentages of CD63+Mito-GFP+ MDRC-derived exosomes, ARF6-Mito-GFP+ MDRC-derived exosomes and individual percentages of CD63+, CD9+ and CD81+ MDRC-derived exosomes (no statistical significance observed; Student's T-test). (e) Histogram and scatter plots of exosomes isolated from MDRCs transduced with Mito-GFP and then labeled with MitoT-Red (n = 3 healthy, n = 3 asthmatics; 3 technical replicates each) (f) Quantitation of the MDRC-derived Mito-GFP+ exosomes labeled ex vivo with MitoT-Red. A significant fraction of exosomes generated were positive for both Mito-GFP and MitoT-Red. (**** p < 0.0001; Two-way ANOVA) (g) Quantitation of the mean fluorescent intensity (MFI) of the MDRC-derived Mito-GFP+ exosomes labeled with MitoT-Red ex vivo. MDRC-derived Mito-GFP+ exosomes from asthmatics had significantly higher MFI of MitoT- Red and Mito-GFP than those derived from MDRCs of healthy subjects (*** p < 0.001, **** p < 0.0001; Two-way ANOVA).