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. 2020 Nov 25;8(2):e001513. doi: 10.1136/jitc-2020-001513

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© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Feasibility of the humanized mouse model for long-term immune monitoring. (A) The proportion of cells in the peripheral blood was analyzed for the humanization marker hCD45+ by flow cytometry over 12–45 weeks from the injection of hCD34+ HSCs. Percentages of hCD45+ cells are demonstrated at three time points (starting point, highest point and final point) according to the myeloablative method or cord blood status. P values were calculated by Student’s t-test. Data are presented as mean±SD. (B) Kaplan-Meier survival curve of humanized mice. Survival of humanized mice was monitored up to 45 weeks according to the myeloablative method and cord blood status (upper). Survival curve was represented according to the myeloablative method (lower). P value was calculated by Breslow (Generalized Wilcoxon) test. (C) Gating strategy for flow cytometry analysis (left). Various immune cell markers, including hCD45, hCD3, hCD4, hCD8, hCD19 and hCD56 in the peripheral blood were analyzed by flow cytometry over 12 weeks to 45 weeks from the injection of hCD34+ HSCs by myeloablative method (right). When hCD45+ cells were taken as a whole (100%), percentages of hCD4+ or hCD8+ cells were generated from hCD45 and hCD3 double positive cells, whereas percentages of hCD19+ or hCD56+ cells were generated from hCD45+ and hCD3− cells. P values were calculated by Student’s t-test. Data are presented as mean±SD. (D) Tumorigenicity test of MDA-MB-231 cells in 45-week-old humanized mice. MDA-MB-231 cells were xenografted at 45 weeks post engraftment of hCD34+ HSCs. Mice were sacrificed 43 days after the implantation of MDA-MB-231 cells; the resultant tumors are demonstrated according to the myeloablative method and cord blood status. Scale bars, 5 mm. (E) Immunohistochemical staining of hCD8 and hCD4 in MDA-MB-231 xenograft tumors of 45-week-old humanized mice. Human tonsil was used for the positive control and non-humanized mouse tumor tissues were used for negative control. H&E images were collected at ×200 magnification and immunohistochemical images were collected at ×400 magnification. The bar graph represented the average of hCD8+ and hCD4+ cells in five random, non-overlapped fields at ×400 magnification. Data are presented as mean±SD.