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. 2018 Dec 6;9:5206. doi: 10.1038/s41467-018-07548-5

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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 — Engrafted brain macrophages display clonal expansion. a Fate-mapping scheme for donor “Microfetti” BM cells in lethally irradiated WT hosts. A single dose of TAM was applied at 2 or 10 weeks after BM reconstitution. BM chimeras were analyzed at 30 weeks after BM transplantation. b Representative sagittal brain section indicating the fields of view (rectangles) analyzed for IBA-1 (magenta) expressing graft microglia in the olfactory bulb (ob), cortex (cx), hippocampus (hc), and cerebellum (cb). DAPI (blue). Scale bar, 1 mm. c–f Representative images of Confetti⁺ (yellow/ red) Iba-1⁺ (magenta) donor cells (arrowheads) in the (c) olfactory bulb (higher magnification in grayscale, inset), (d) cortex, (e) hippocampus, and (f) cerebellum. DAPI (blue). Scale bars, 50 µm (c–e) and 100 µm (f). g–j Frequency of Confetti⁺ IBA-1⁺ graft as single cells or clones in analyzed fields from TAM treatment groups of 2 (white) and 10 weeks (gray) after BM transplantation. Each dot represents the mean quantification of one animal. At least four sections per animal were analyzed. Mann–Whitney test, *P = 0.0357, 0.0179, respectively in (j). k Representative images of cortical eBFP⁺ (white) IBA-1⁺ (red) graft-derived cells in brains of TBI and busulfan-conditioned mice. DAPI (blue). Higher magnification in insets. Scale bars, 50 µm. l Representative barcode analysis on DNA extracted from peripheral blood, bone marrow, spleen, and sorted CD45^int brain macrophages and CD45^high cells of a busulfan- and a TBI-conditioned mouse. Barcode numbers for each sample are shown in the bar plot and the amount of shared barcodes per sample are displayed in the Venn diagrams. m Number of barcodes detected in engrafted macrophage samples of TBI- and busulfan- conditioned animals. Each dot represents one animal (n = 4 per group). n Bar graph showing percentages of barcodes private to grafted cells among CD45^int and CD45^hi CNS cells (i.e., parenchymal and non-parenchymal macrophages, respectively), as well as BM cells of TBI- and busulfan-conditioned animals (n = 4 per group)