应用双光子显微镜和流式细胞仪定性及定量确定小鼠巨噬细胞的吞噬功能

建立了流式细胞仪和双光子激光共聚焦荧光显微镜进行定性和定量检测小鼠巨噬细胞吞噬鸡红细胞的方法,并同传统光学显微镜细胞化学染色观察方法相比较,探讨其检测巨噬细胞吞噬效应的优越性。常规方法获取小鼠腹腔和脾脏巨噬细胞,制备巨噬细胞悬液。常规制备鸡红细胞,计数并调整活细胞数,用5-二醋酸羧基荧光素琥珀酸单胞菌酯(5-carboxyfluorescein diacetate succinimidyl ester,CFSE)染色,与巨噬细胞共温育一定时间后,小鼠巨噬细胞特异性荧光抗体F4/80标记巨噬细胞。应用流式细胞仪检测巨噬细胞中CFSE阳性百分率来表示巨噬细胞吞噬率;应用双光子显微镜观察被吞噬的CFSE阳性鸡红细胞动态分布情况。同时,采用传统光学显微镜吉姆萨染色观察巨噬细胞吞噬百分率。结果显示,流式细胞仪结合双光子显微镜检测巨噬细胞吞噬率与传统的显微镜计数法比较,两者有明显的正相关性。双光子显微镜和流式细胞仪可以定性与定量检测巨噬细胞吞噬功能,该方法具有灵敏、快捷、重复性好以及准确率高的特点,是进行免疫学研究的可行方法。

The Phagocytosis Ability of Mouse Macrophages Determined by Flow Cytometry and Two Photon Microscope

The objective of this study is to establish an approach to determine the chicken red blood cell phagocytosis ability and process of mouse macrophages by a flow cytometry (FCM) and a two photon microscope (TPM), as well as to investigate the advantages of the new method over the traditional cytochemical staining and light microscope method. Mouse peritoneal lavage and splenic macrophage samples were prepared. Chicken red blood cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). The macrophages were incubated with the chicken red blood cells for proposed time. Macrophages were stained with PE-anti-F4/80 monoclonal antibody. The phagocytosis rates of CFSE-positive cells were determined by FCM and the phagocytized chicken red blood cell distribution in macrophages were observed by TPM. Meanwhile, macrophage phagocytosis was observed by traditional Giemsa staining and light microscope method. The macrophage phagocytosis measured by FCM and TPM or the traditional method showed similar results. In addition, the measurement of the phagocyto-sis activity of macrophages by FCM and TPM is sensitive, quick, accurate and of good reproducibility. The present approach would be helpful for us to perform macrophage immunology research.