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. 2019 Oct 1;9:14050. doi: 10.1038/s41598-019-50320-y

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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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Flow cytometry analysis of liver samples from Ehrlichia-infected mice. (A) Flow cytometry gate strategy for the analysis of liver samples. Ungated events were initially plotted as FSC-A × FSC-H for exclusion of cell aggregates located far from the main diagonal. Considered cells were then morphologically represented in SSC × FSC density plots where a region containing mainly CD11b⁺ and F4/80⁺ cells (as confirmed by back gating) was defined. From these considered events, CD3-positive cells were excluded and the resultant events were plotted as CD11b × F4/80 for the analysis of infiltrating macrophages/monocytes defined as F4/80^loCD11b^hi cells, and the Kupffer cells, considered as F4/80^hiCD11b^lo cells. Values represent frequencies in the assigned regions. (B) Each of the considered macrophage populations was also addressed for the intracellular expression of iNOS and granzyme B (Grz B) and represented as flow histograms. Values represent frequencies in the assigned regions. (C) Differential expression of CD68 between non-resident macrophages and Kupffer cells. Flow cytometry dot plot representative of an E. muris-infected animal exhibiting the considered regions for Kupffer cells (F4/80^hiCD11b^lo cells) and non-resident or infiltrating macrophages/monocytes (F4/80^loCD11b^hi cells). Each of these regions were checked for the expression of CD68 and as expected Kupffer cells are positive and non-resident cells are negative as shown by the flow histogram overlay. This observed pattern of CD68 expression held for every analyzed groups, naive, E. muris (EM) and Ixodes Ovatus Ehrlichia (IOE). Events in the blue peak are gated in F4/80^hiCD11b^lo region and events from the red peak are gated on the F4/80^loCD11b^hi region. (D) Quantification of the analyzed cell sub-populations in the different studied groups. Values are expressed as mean and standard deviation of percentage. Asterisks represent relevant statistical difference between groups. Data is representative of three experimental sets performed individually with n = at least three mice per group in each experimental run.