Demonstration of B-cell maturation in X-linked immunodeficient mice by simultaneous three-colour immunofluorescence

CBA/N mice carrying the X-linked immune deficiency gene (xid) have fewer splenic B cells than normal CBA mice and are unresponsive to a certain class of antigens. Studies of B-cell surface-marker expression and immune responsiveness have led to the commonly accepted idea that the B cells in adult xid mice are immature and resemble the B cells of young (1-3 week old) normal mice. That is, like young animals, xid mice lack cells in the most numerous of three IgM/IgD B-cell subpopulations (designated I in Fig. 1a, b) present in adult spleen. We now report, however, that this picture is an oversimplification and that in fact the B cells in adult xid mice differ from those present in either adult or young normal mice. Using quantitative three-colour fluorescence-activated cell sorter (FACS) analyses, we have compared the correlated expression of IgM, IgD and a newly discovered B-lymphocyte antigen (BLA-1) on splenic B cells in normal and xid mice. We show here (1) that most B cells in adult xid mice (as in normals) are BLA-1- whereas all B cells in young animals are BLA-1+; (2) that the major difference in the IgM/IgD B-cell subpopulations found between xid and normal mice is limited to the BLA-1- cells; and (3) that xid mice have increased numbers of BLA-1+ population III B cells.     

基于磁分选技术和间接免疫荧光技术的肺癌A549细胞的分离检测

采用磁分选技术及间接免疫荧光技术分离检测肺癌循环肿瘤细胞(CTCs).将表皮生长因子受体(EGFR)抗体连接到四氧化三铁(Fe3O4)磁珠表面构建免疫磁珠(MNPs-Anti-EGFR),通过EGFR抗体和肺癌A549细胞的表面抗原的特异性结合,将免疫磁珠与肺癌细胞结合.通过磁场作用,使肺癌细胞在磁场的作用下从混合细胞中分离富集.分离出的细胞利用间接荧光技术在其细胞表面连接结合PE荧光团的特异性抗体,并用4',6-二脒基-2-苯基吲哚(DAPI)对细胞核染色以定位细胞位置.通过倒置荧光显微镜的观察,从而实现对肿瘤细胞的定量计数检测.实验结果显示该方法对肺癌A549细胞具有较好的检测灵敏度和检测下限,检测下限可达3 cells/m L.