INTRODUCTION OF UNLABELED PROTEINS INTO LIVING CELLS BY ELECTROPORATION AND ISOLATION OF VIABLE PROTEIN-LOADED CELLS USING DEXTRAN FLUORESCEIN ISOTHIOCYANATE AS A MARKER FOR PROTEIN-UPTAKE
INTRODUCTION OF UNLABELED PROTEINS INTO LIVING CELLS BY ELECTROPORATION AND ISOLATION OF VIABLE PROTEIN-LOADED CELLS USING DEXTRAN FLUORESCEIN ISOTHIOCYANATE AS A MARKER FOR PROTEIN-UPTAKE
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DOI：
10.1016/0003-2697(91)90168-s
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发表时间：
1991-04-01
影响因子：
2.9
通讯作者：
BARSAGI, D
中科院分区：
文献类型：
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作者：
GRAZIADEI, L;BURFEIND, P;BARSAGI, D
通常，显微注射是将蛋白质引入活细胞的首选方法。然后可以鉴定含有引入蛋白的活细胞，只要该蛋白是荧光染料偶联的。这种方法只适用于贴壁细胞，而且可以分析的细胞数量很少。在这项研究中，我们已经确定了电穿孔可以用于将蛋白质高效地装载到大量细胞中。此外，我们还开发了一种分离蛋白质负载细胞的方法，使用荧光素异硫氰酸酯-葡聚糖（dextran-FITC）作为蛋白质摄取的分子标记。该方法的基本特点是右旋糖酐- fitc包含在电穿孔介质中，因此，与感兴趣的蛋白质共同引入。使用荧光激活的细胞分选纯化含有葡聚糖- fitc的细胞，产生的群体几乎完全由含有目标蛋白的细胞组成。
Commonly, microinjection has been the method of choice for introducing proteins into living cells. Viable cells containing an introduced protein can be then identified providing that the protein is fluorochrome conjugated. This approach is applicable only for adherent cells, and the number of cells that can be analyzed is small. In this study, we have established that electroporation can be used to load proteins into large numbers of cells with high efficiency. Furthermore, we have developed a method for the isolation of protein-loaded cells using fluorescein isothiocyanate—dextran (dextran—FITC) as a molecular marker for protein uptake. The essential features of this method are that dextran—FITC is included in the electroporation medium and, thus, is cointroduced with the protein of interest. Purification of cells containing dextran—FITC using fluorescence-activated cell sorting yields a population which is composed almost entirely of cells containing the protein of interest.