Abstract

Spleen is an information-rich and easy-accessible peripheral lymphoid organ. It has complex cell composition because of the immunocytes maturity and settle down. Changes of the composition and function of these immunocytes are critical to body immune response. To understand the cell behaviors, specific cell subpopulations are required to be separated without heterogeneity. Density gradient centrifugation is one of the cell separation methods with high throughput. However, the greatest defect of this method is its low cell purity. In this study, the separation conditions of tumor-bearing mouse splenocytes were optimized by separation solutions with different density gradients. After separation, lymphocytes were located at the second layer with the proportion of 84.9%, monocytic-like myeloid-derived suppressor cells (Mo-MDSCs) were located at the fourth layer with the proportion of 54.2% and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were located at the sixth layer with the proportion of 85.5%. Cells in different layers were further determined by verifying the gene expression pattern of some chemokine receptors on cell surfaces. Furthermore, this method was also used to separate healthy mouse splenocytes. Therefore, this method will be highly useful to separate mouse splenocytes and has laid a foundation for further research on the changes and roles of immunocytes during the development of cancer.

摘要

脾脏是一种信息丰富且易于访问的外周淋巴器官。由于免疫细胞的成熟和稳定，它具有复杂的细胞组成。这些免疫细胞的组成和功能的变化对身体免疫反应至关重要。要了解细胞行为，需要在没有异质性的情况下分离特定的细胞亚群。密度梯度离心是一种高通量的细胞分离方法。但是，该方法最大的缺陷是细胞纯度低。本研究通过不同密度梯度的分离液对荷瘤小鼠脾细胞的分离条件进行了优化。分离后，淋巴细胞位于第二层，占 84.9%，单核细胞样髓源性抑制细胞（Mo-MDSCs）位于第四层，比例为54.2%，多形核髓源性抑制细胞（PMN-MDSCs）位于第六层，比例为85.5%。通过验证细胞表面一些趋化因子受体的基因表达模式，进一步确定了不同层的细胞。此外，该方法还用于分离健康的小鼠脾细胞。因此，该方法对于分离小鼠脾细胞将非常有用，并为进一步研究免疫细胞在癌症发展过程中的变化和作用奠定了基础。通过验证细胞表面一些趋化因子受体的基因表达模式，进一步确定了不同层的细胞。此外，该方法还用于分离健康的小鼠脾细胞。因此，该方法对于分离小鼠脾细胞将非常有用，并为进一步研究免疫细胞在癌症发展过程中的变化和作用奠定了基础。通过验证细胞表面一些趋化因子受体的基因表达模式，进一步确定了不同层的细胞。此外，该方法还用于分离健康的小鼠脾细胞。因此，该方法对于分离小鼠脾细胞将非常有用，并为进一步研究免疫细胞在癌症发展过程中的变化和作用奠定了基础。