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. 2019 Jun 20;10:2699. doi: 10.1038/s41467-019-10587-1

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© The Author(s) 2019

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Physical attributes of ARPE-19 cell-derived vesicles and their humoral immune response in mice. a Increasing amounts of ARPE-19 membrane fraction (μg) were resolved on a SDS-polyacrylamide gel (12.5%) and visualized using Gel Code Blue Reagent (ThermoFisher Scientific). b Transmission electron microscopy (TEM) images of the membrane fraction. Representative images demonstrate the range in vesicle sizes within the fraction. Arrows indicate observed protein aggregates surrounding vesicles. c The distribution of vesicle diameters (nm) (n = 29). Bounds of the box span 25–75% percentile, the center line represents median, and whickers visualize 5 and 95% of the data points. d Dilutions of serum from five mice (M1–M5) immunized with the ARPE-19 membrane fractions were subjected to flow cytometry with ARPE-19 cells. Normal mouse serum (NMS), PBS, and the mAb W6/32 (targeting MHC class I) were used as controls. e Two dilutions of serum from the ARPE-19 vesicle immunized mice were subjected to a high-throughput infectivity assay (HTI) with TB40/E^{FLAG YFP} infection of ARPE-19 cells using YFP fluorescence as readout for infection. The % infection was determined using virus incubated with normal mouse serum (NMS) as 100%. Infection data was performed in triplicate. s.d. is depicted in the experiment. **P < 0.01(Student’s two-tailed t test)