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. 2021 Dec 25;11:220. doi: 10.1186/s13578-021-00732-z

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Characterization of Sucrose Gradient-Purified Exosomes. A Exosomes from both RVFV-infected cells and uninfected cells were harvested in parallel using the procedure describe in the Materials and Methods section. The sucrose density fractions were initially analyzed by western blot for the exosome marker CD63 (top). CD63-positive fractions with densities 1.08 g ml^-1 and 1.12 g ml^-1 were combined and further analyzed by western blot for additional exosome markers TSG101 (middle) and Flotillin-1 (bottom); three independent preparations were tested and are denoted on the figure as “Sample A”, “Sample B”, and “Sample C”. WCL refers to U937 whole cell lysate used as positive control. At least 25 biological replicates of this purification scheme have been performed and analyzed; the data presented here are representative of the findings. B Mean values ± SEM of plaque assay results from three biologically independent EXi-RVFV preparations are shown, quantifying the virions present in the starting material and following the ultracentrifugation spins and sucrose gradient separation. Plaque assays were performed for every single purified exosome preparation throughout our studies, totaling at least 15 biological replicates. A summary of plaque assay results for all the fractions is also presented. The red box highlights the two fractions that were combined to generate EXi-RVFV used in our studies (1.08 g ml-1 and 1.12 g ml-1 fractions). Circles with blue color in the middle represent exosomes and circles with yellow color in the middle represent virions. C TEM analysis of purified exosomes is shown. D ZetaView analyses of the mean diameter of the EXu and EXi-RVFV populations are presented. Three biological replicates were analyzed. E ZetaView measurements of the surface charge (Zeta Potential) for EXu and EXi-RVFV samples are presented. Mean values ± SEM from three biological replicates are shown. F ZetaView measurements of the concentration of EXu and EXi-RVFV samples that were matched for total protein content based on BCA are presented. Mean values ± SEM from three biological replicates are shown