Electroporation of single cells and tissues with an electrolyte-filled capillary.
Electroporation of single cells and tissues with an electrolyte-filled capillary.
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使用充满电解质的毛细管对单个细胞和组织进行电穿孔。
DOI：
10.1021/ac010403x
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发表时间：
2001
影响因子：
7.4
通讯作者：
Orwar,O
中科院分区：
文献类型：
--
作者：
Nolkrantz,K;Farre,C;Brederlau,A;Karlsson,RI;Brennan,C;Eriksson,PS;Weber,SG;Sandberg,M;Orwar,O
我们展示了如何将电解质填充的毛细管（EFC）耦合到高压电源，可用作将染料，药物和生物分子递送到组织中的单细胞和细胞的细胞质的多功能电穿孔工具。在EFC（熔融石英，30 cm长，375 μm o.d.，30 μm i.d.）在EFC的末端外产生小电场，这导致细胞膜中的孔形成并诱导电解质的电渗流。当EFC含有细胞负载剂时，电渗流将试剂递送到孔形成部位。孔形成和递送的组合使得能够将材料加载到细胞质中。通过膜片钳和荧光显微镜，在估计的<85 mV的跨膜电压下观察到孔的形成，半最大值约为206 mV。通过将荧光染料引入单个NG 108 -15细胞、细胞过程和器官型海马培养物中的小细胞群体来证明电穿孔方案。初步结果显示，该协议是用于在大鼠脑腹侧中脑的体内电穿孔。该技术还用于访问细胞内基于细胞器的检测系统。作为示范，将1，4，5-肌醇三磷酸添加到电解质中并通过电穿孔细胞中的细胞内细胞器检测。
We show how an electrolyte-filled capillary (EFC) coupled to a high-voltage power supply can be used as a versatile electroporation tool for the delivery of dyes, drugs, and biomolecules to the cytoplasm of single cells and cells in tissues. A large-voltage pulse applied across the EFC (fused silica, 30 cm long, 375-μm o.d., 30-μm i.d.) gives rise to a small electric field outside the terminus of the EFC, which causes pore formation in cell membranes and induces an electroosmotic flow of electrolyte. When the EFC contains cell-loading agents, then the electroosmotic flow delivers the agents at the site of pore formation. The combination of pore formation and delivery enables loading of materials into the cytoplasm. By patch-clamp and fluorescence microscopy, formation of pores was observed at estimated transmembrane voltages of <85 mV with half-maximum values around 206 mV. The electroporation protocol was demonstrated by introduction of fluorogenic dyes into single NG108-15 cells, cellular processes, and small populations of cells in organotypic hippocampal cultures. Preliminary results are shown in which this protocol was employed for in vivo electroporation of ventral mesencephalon in rat brains. The technique was also used to access organelle-based detection systems inside cells. As a demonstration, 1,4,5-inositoltriphosphate was added to the electrolyte and detected by intracellular organelles in electroporated cells.