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. 2018 Aug 10;9:3194. doi: 10.1038/s41467-018-05211-7

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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 — Cell surface CRT determines programmed cell removal of neutrophils in peritonitis. a, b Cell surface levels of CRT on macrophages and neutrophils after thioglycollate injection to MRP8-Bcl2 mice. Peritoneal cells were collected and analyzed at 0, 4, 6, 12, and 24 h after thioglycollate injection, by flow cytometry analysis. Results are representatives of three independent experiments. n = 3 mice for each time points. *P < 0.05 (paired t-test) for CRT staining between macrophages and neutrophils at the same time points. Error bars represent standard deviation. b Cell surface levels of CD47 on macrophages and neutrophils after thioglycollate injection to MRP8-Bcl2 mice. Peritoneal cells were collected and analyzed at 0, 4, 6, 10, and 24 h after thioglycollate injection, by flow cytometry analysis. Results are representatives of three independent experiments. n = 3 mice for each time point. n.s. (paired t-test) so significant differences for CD47 staining between macrophages and neutrophils at the same time points. Error bars represent standard deviation. c Binding of recombinant CRT to macrophages and neutrophils after thioglycollate injection to MRP8-Bcl2 mice. Peritoneal cells were collected and analyzed at 0, 4, 6, 10, and 24 h after thioglycollate injection. CRT-binding sites on macrophages and neutrophils were measured by incubating the cells with saturation concentration of recombinant CRT and analyzing by flow cytometry. Results are representatives of three independent experiments. n = 3 mice for each time point. *P < 0.05 (paired t-test) for rCRT staining between macrophages and neutrophils at the same time points. Error bars represent standard deviation. d An in vitro phagocytosis assay showing blockade of CRT inhibits phagocytosis of neutrophils, with WT and Bcl2 neutrophils as target cells and peritoneal macrophages. Neutrophils were collected 4 h after thioglycollate treatment and cultured for 24 h. Phagocytosis was normalized to the maximal response in the experiments. n = 3. **P < 0.01 (t-test) for phagocytosis between ctrl and CRT blocking Ab treatment. Error bars represent standard deviation. In a, b, and c, MFI, mean fluorescence intensity