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. 2019 Nov 7;14:8755–8768. doi: 10.2147/IJN.S209366

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© 2019 Ferrantelli et al.

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Detection of fluorescent exosomes in challenged SiHa cells. (A) On the left: fluorescence microscope analysis of HEK293T cells transfected with Nef^mut/GFP vector. On the right, the same analysis was carried out on mock-transfected SiHa cells. The cell nuclei were stained with DAPI (blue fluorescence). (B) FACS analysis of fluorescent exosomes produced by Nef^mut/GFP expressing cells. Exosomes were bound to surfactant-free white aldehyde/sulfate latex beads and then labeled with PE-conjugated anti-CD63 mAbs. The analysis included singularly dispersed beads only, as identified through the FSC/SSC plots. Shown are the results representative of six independent experiments. (C) Western blot analysis of 2 mU of exosomes purified from the supernatants of Nef^mut/GFP expressing cells. As control, exosomes from cells transfected with the vector expressing Nef^mut/43M2 were included. Polyclonal anti-Nef Abs served to detect Nef^mut-based products, while Alix was revealed as exosome marker. Molecular weight markers are given in kilodaltons (kDa). The results are representative of two independent experiments. (D) FACS analysis on SiHa cells challenged with GFP-fluorescent exosomes. A total of 2×10⁴ SiHa cells was challenged with different amounts of fluorescent exosomes (i.e., from 80 to 240 μU). After spinoculation, cells were incubated either at either 4°C or 37°C. At different times, cells were detached with cold trypsin, washed, fixed, and FACS analyzed. On the top, shown are the raw data from cells challenged or not (Mock) with 160 μU of exosomes, and analyzed 3 hrs later. As control, the results from exosome-challenged cells incubated at 4°C are shown. On the bottom left, shown are the results from FACS analysis of cells challenged with 160 μU of exosomes and harvested at different times. As control, conditions where exosome-challenged cells were incubated at 4°C and harvested at the latest time point were included. On the bottom right, shown are the results from FACS analysis of cells challenged with the indicated amounts of fluorescent exosomes, and harvested 3 hrs after spinoculation. As control, data from the analysis of cells challenged with the highest amounts of exosomes and incubated at 4°C were included. The results are presented as mean values +SD of triplicates after subtraction of background values (i.e., the fluorescence levels of mock-challenged cells), and are representative of two independent experiments.