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本文作者建立了一种能够检测出产生抗HLA抗体的单个细胞的空斑试验。试验原理是用绿色荧光素(碳氧荧光素乙酰乙酸盐,CFDA)染活细胞,红色荧光素(Propidum iodide PI)染空斑内由抗体和补体作用溶解的细胞,在荧光显微镜下计数。标本干燥后可长期保存而不发生退色等改变。空斑形成的主要原理是细胞溶解,靶细胞释放荧光素,摄取PI而形成空斑。镜下可见桔红色的斑点,在其周围有带绿色荧光素的细胞围绕。靶细胞的来源是经EB病毒刺激后转化的淋巴母细胞。该细胞在加有L-谷氨酰胺和热灭活10%胎牛血清RPMI 1640培养液中培养,在其对数生长期收获,再用CFDA进行染色。抗体分泌细胞是通过鼠—鼠杂交瘤
The authors established a plaque assay that detects single cells that produce anti-HLA antibodies. The principle of the experiment was to stain the living cells with green fluorescein (CFU), Propidum iodide PI, and to stain the cells dissolved by the antibody and complement in the plaque and count under a fluorescence microscope. Specimens can be stored for long periods of time after drying without fade or other changes. The main principle of plaque formation is cell lysis, release of fluorescein by target cells, uptake of PI and formation of plaques. Microscopic orange-red spots were visible around the cell with green fluorescein. The source of target cells is lymphoblastoid cells that have been transformed by the Epstein-Barr virus. The cells were cultured in RPMI 1640 medium supplemented with L-glutamine and heat-inactivated 10% fetal bovine serum, harvested at their logarithmic growth phase, and stained with CFDA. Antibody-secreting cells are made through murine-mouse hybridomas