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. 2018 Jun 15;9:2359. doi: 10.1038/s41467-018-04791-8

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© The Author(s) 2018

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. 2 — Electroporation of RBCEVs with ASOs and delivery to leukemia cells. a Experimental scheme of ASOs delivery by RBCEVs. b Average concentration of EVs (200× dilution) and average fold change in FAM fluorescent intensity relative to unelectroporated EVs (UE-EVs) of 12 fractions in a sucrose gradient separation of RBCEVs electroporated with a FAM-labeled scrambled negative control ASOs (FAM-NC-ASOs), determined using a Nanosight analyzer and Synergy fluorescent microplate reader, respectively, n = 3 repeats. c Separation of unbound NC-ASOs (unlabeled) from 8.25 × 10¹¹ unelectroporated or electroporated RBCEVs compared to the untreated NC-ASOs (200 pmol) in 10% native gel, visualized using SYBR Gold staining (top) and the average percentage of NC-ASOs unbound or bound to electroporated RBCEVs (bottom), n = 3 independent replicates. d FACS analysis of FAM fluorescence vs. forward scatter area (FSC-A) in MOLM13 cells transfected with 400 pmol FAM-NC-ASO using LipofectamineTM 3000 (Lipo), INTERFERin^® (Inte) or 12.4 × 10¹¹ RBCEVs. Percentages of FAM-positive cells are indicated above the gate. e Average percentages of FAM⁺ cells among viable MOLM13 cells transfected or treated with RBCEVs containing FAM ASOs as in d, n = 3 repeats. f Percentages of dead cells determined by propidium iodide staining among MOLM13 cells transfected or treated with RBCEVs containing unlabeled NC-ASOs, n = 4 repeats. All graphs present mean ± SEM. Student’s one-tail t-test results are shown as n.s. non-significant; **P < 0.01; ***P < 0.001 and ****P < 0.0001 relative to the unelectroporated control (c) or to the untreated control (e, f)