The mouse xenograft model is one of the most widely used animal model for biomedicine research. It is vital to distinguish the cells from different species, especially for the spatial distribution information. However, the available strategies of species-specific detection are either inapplicable in situ or of low specificity. Here, we reported a method based on DAPI staining, which offers an effective, convenient way that accurately identifies human and mouse nuclei at single-cell level in situ. This method was proven to be effective in cell co-culture and tumor xenograft tissue section. Microscopic imaging results shows obvious DAPI plaques-like structures in mouse nuclei, but absent in human nuclei. Moreover, we found these structures are co-localized with mouse major satellite DNA, which is located pericentromere in mouse, but absent in human.Our study provides a high-performance method that can be widely used for distinguish human and mouse cell in situ.#33342 (data not shown) and histone (GFP-H2B) Specific fluorescent labeling (Kanda, Sullivan et al., 1998); Supplementary Figure 4), etc. In the future, the aspects related to human and mouse cells can be considered for further application through this method, such as drug development, cell identification, identification of true and false fluorescent pictures, etc.
Methods
Cell cultureBoth mouse and human cell lines used in this work were obtained from the American Type Culture Collection (ATCC), and cultured in DMEM or F12 medium supplemented with 10% FBS.Macrophages were obtained by referring to literature and cultured in complete medium containing serum (Baghdadi, Wada et al., 2016; Weischenfeldt & Porse, 2008). All the cells were cultured at 37 ° C humidified incubator containing 5% CO 2 .
Mouse model and tissue acquisitionLocal tumor tissues removed from breast tumor patients were used to establish a mouse model of PDX. The patient's written informed consent was obtained in advance, and the research protocol was approved by the hospital ethics committee. All experiments used immunodeficient mouse were performed according to guidelines approved by the Institutional Animal Health and Use Committee (IACUC). Fresh surgical tumor tissues were implanted directly into immunodeficient mouse to establish a PDX mouse model, and SNP48 analysis confirmed that the established PDX was successful. In the breast tumor cell line transplant model, we used MCF-7 cells. The cultured cells were implanted directly into the skin of immunodeficient mouse to establish a CDX mouse model. In the orthotopic transplantation model of liver tumor cell lines, we used Huh7 cells, and the cultured cells were directly implanted into the liver of immunodeficient mouse in situ to establish a mouse model. After tumor masses were formed in the above mice, they were extracted and embedded in paraffin for sectioning.
DNA fluorescence in situ hybridizationThe prepared cell and tissue samples were fixed with 4% formaldehyde for 10 minutes, and then washed in PBS-T for 30 minutes. The fixed sample...
