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. 2021 May 5;11:9529. doi: 10.1038/s41598-021-88897-y

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© The Author(s) 2021

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 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/.

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Characterization of microvesicles (MVs) produced from pneumolysin (PLY)-treated alveolar epithelial cells. A549 cells were treated with PLY (100 ng/ml). After 4 h, conditioned media was collected and MVs were isolated and characterized. (A,B) Representative FACS analysis (overlay dot blots) of isolated vesicles (blue dots), and of (A) 1 μm beads (red dots) or (B) human platelets (green dots). (C) MVs were stained with annexin V-APC in the presence or absence of calcium and analyzed by FACS. (D) MVs were treated with Triton-X 100 and analyzed by FACS. (E) CFSE-labeled MVs (green) were visualized using confocal microscopy. (F) MVs were labeled with CFSE (FITC) and CellMask (APC) and analyzed by FACS. In the representative dot blot, MVs labeled with CFSE and CellMask are shown in green. Grey dots represent unstained vesicles (background staining).