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. 2022 Sep 9;8:89. doi: 10.1038/s41421-022-00453-8

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© The Author(s) 2022

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 — a Imaging flow cytometry analysis of cytoplasmic and surface ACE2 protein in CD14⁺ MO with or without R848 treatment up to 4 h. Each cell is represented by the row of images that include bright field (BF), DAPI (purple), CD14 (turquoise), ACE2 (red), and the overlapping image merged with DAPI, CD14, and ACE2. b Histograms of ACE2 intensity on the cell surface of CD14⁺ monocytes treated with R848 (left). Grey line represents the isotype control group (Iso). Column graphs indicate MFI of ACE2 surface intensity at other indicated conditions (right) (n = 3 biologically independent samples/group). c Representative confocal microscopy of ACE2 protein in CD14⁺ monocytes with or without R848 treatment for 24 h. Bars, 10 µm. Dashed lines show location of cell membrane. Images show cells from two healthy donors with similar results. d Immunoblot showing ACE2 and CD63 expression in exosomes isolated from plasma of healthy donors. The monkey kidney epithelial cell line Vero was used as a positive control. e Flow cytometry analysis of ACE2 surface expression in PBMCs pre-treated with the indicated concentrations of endosidin 2 (ES2) or DMSO for 1–2 h before ex vivo incubation with R484, LPS, or medium alone for 4 h. f Frequency of ACE2⁺ MO for data shown in e. Bars represent means ± SEM of biologically independent samples (n = 3/group for 40 µM ES2 condition; n = 4/group for all other conditions).