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. 2020 Mar 13;10:4681. doi: 10.1038/s41598-020-61679-8

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© The Author(s) 2020

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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Determination of optimal cell number during development of the metastatic mouse model. (A) Representative photograph of injection of 0.2% trypan blue cells into the mammary glands of mice. (B) Representative photograph of the whole mount of the 4th inguinal mammary glands of female BALB/C mice injected with 20,000 4T1 cells for 1 week (n = 5). LN: lymph node. (C) Representative photograph of the whole mount of the 4th inguinal mammary glands of female BALB/C mice injected with 4T1 cells for 2 weeks (n = 5). LN: lymph node. (D) Rate of tumor formation and time of tumor formation after injection with different numbers of cells. ns. not significant, ****p < 0.0001. (E) Consequences of different numbers of injected cells.