Screening Antibodies With Cross-reactivity To Immune Cells Of Tree Shrew And Immunophenotypic Analysis

Objective:Tupaia belangeri,a kind of mammal,belongs to the Scandentia,family Tupaiidae.The experimental results from tree shrews are easy to be translated to human medical biology research.Scientists have successfully domesticated and nurtured a number of tree shrews,widely used in the establishment of disease models.At the same time,a series of techniques were established for tree shrews as experimental animals.The immune system plays a very important role in the body's resistance to all kinds of microorganisms,toxins and carcinogens in the external environment,and evaluation of immune responses is an indispensable part of the application of animal models.The lack of knowledge about the immune system of tree shrew leads to difficulties in studying the pathogenesis of the disease in depth,and further developing measures for the disease prevention and control.Therefore,characterization of the immune system of tree shrews is very important for their development as experimental animals.In this study,we analyzed the phenotype of immune cells of tree shrew.Methods:1.Four reagents of RBCs lysis were tested and compared for the PB of tree shrew,followed by optimization of protocols and conditions for RBCs lysis,in order to obtain an optimized condition that gives effective lysis of RBCs and mostly viable cells for immunophenotypic analysis by flow cytometry.2.Based on the cross-reactivity of antibodies against a particular antigen,we screened a libraray of commercially available antibodies on immune cells of tree shrew.We analyzed the cross-reactivity of the antibodies by flow cytometry in order to identify the most reactive antibody clone.3.The antibodies with cross-reactivities were used to further analyze the phenotypes of immune cells from tree shrew.By flow cytometry,the percentages of various types of cells were determined and compared among differnet genders and ages of tree shrews.Results:1.The results of optimization of RBCs lysis showed that 10 times of lysis buffer over the whole blood volume for lysis,two lyses(10 minutes and 5 minutes)performed in a volume of 300 ?l led to the maximal lysis of RBCs and the best viability of leukocytes in the PB of tree shrew.2.Form the 98 commercially available antibodies,we found that 74 antibodies showed relative cross-reactivity with the immune cells of peripheral blood in tree shrews,65 antibodies showing cross-reactivity with immune cells in bone marrow,and 62 antibodies cross-reacting with immune cells from spleen.The surface markers recognized by these identified antibodies included CD3,CD4,CD8,CD 19,CD20 and CD45,which may be used for analysis of lymphocytes,macrophages,natural killer cells and other immune cells.3.Primary charaterization of immune cells from tree shrew.By combination of antibodies against various CD markers ofr immune cells,we determined the frequencies of immune cells from tree shrews with different ages or genders by flow cytometric analysis.In peripheral blood of tree shrew,the proportion of CD3+CD4+CD45+ cells in the gate of white blood cell is 2.15%to 8.25%,the CD3+CD8+CD45+ cells with the frequency of 5.80%to 7.64%,the CD19+CD20+CD45+ cells with 3.02 to 9.56%,the CD16+CD56+ cells with 24.19%to 32.72%,the CD11b+CD14+ cells with 3.48%to 15.03%,the CD11b+CD13+CD 16+CD45+ cells with 0.04%to 3.33%.In bone marrow of tree shrew,the proportion of CD3+CD4+CD45+ cells in white blood cells is 2.17%to 6.39%,the CD3+CD8+CD45+ cells with 1.57%to 7.06%,the CD19+CD20+CD45+ cells with 0.41%to 4.70%,the CD 16+CD56+ cells with 14.20%to 32.00%,the CD11b+CD14+cells with 3.46%to 8.70%,the CD11b+CD13+CD16+CD45+ cells with 0.01%to 0.86%,In spleen of tree shrew,the proportion of CD3+CD4+CD45+ cells in white blood cell is 1.37%to 2.15%,the CD3+CD8+CD45+ cells with 5.26%to 12.00%,the CD19+CD20+CD45+ cells with 0.45%to 6.65%,the CD16+CD56+ cells with 13.34%to 18.74%.Conclusion:By opitimization of RBCs lysis and screening the selected antibodies with cross-reactivities to immune cells from tree shrew,we established an effective protocol of RBCs lysis and found a number of cross-reactive antibodies.Finally,we determined the frequencies of immune cells in tree shrew.These results provides a solid theoretical basis and a necessary research platform for immunophenotypic analysis of immune cells and various disease models of tree shrew.