Sczakiel G, Döffinger R, Pawlita M
Institut für Virusforschung, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany.
Anal Biochem. 1989 Sep;181(2):309-14. doi: 10.1016/0003-2697(89)90248-0.
A nonradioactive and rapid method to systematically optimize conditions for electrotransfection is described here for a critical parameter, the initial voltage. This technique utilizes the electric field-dependent transfer of the fluorescent compound Lucifer Yellow CH into cells. Dye uptake can be followed and quantified by fluorescence microscopy for individual cells or in sum by fluorescence spectroscopy. Electrotransfection conditions for maximal dye and DNA uptake correspond with each other. Cotransfection of Lucifer Yellow CH and DNA coding for the indicator gene chloramphenicol acetyltransferase followed by fluorescence-activated cell sorting demonstrates that the cell population that takes up the fluorescent compound also expresses the indicator gene.
本文描述了一种用于系统优化电穿孔转染关键参数——初始电压条件的非放射性快速方法。该技术利用荧光化合物路西法黄CH在电场作用下转入细胞。可以通过荧光显微镜对单个细胞进行染料摄取跟踪和定量，也可以通过荧光光谱对总体进行跟踪和定量。实现最大染料和DNA摄取的电穿孔转染条件相互对应。将路西法黄CH与编码指示基因氯霉素乙酰转移酶的DNA共转染，随后进行荧光激活细胞分选，结果表明摄取荧光化合物的细胞群体也表达指示基因。
