Department of Functional Molecular Biology, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8512, Japan.
J Exp Biol. 2013 Oct 1;216(Pt 19):3591-8. doi: 10.1242/jeb.089870. Epub 2013 Jun 20.
The labeling of specific molecules and their artificial control in living cells are powerful techniques for investigating intracellular molecular dynamics. To use these techniques, molecular compounds (hereinafter described simply as 'samples') need to be loaded into cells. Electroporation techniques are exploited to load membrane-impermeant samples into cells. Here, we developed a new electroporator with four special characteristics. (1) Electric pulses are applied to the adherent cells directly, without removing them from the substratum. (2) Samples can be loaded into the adherent cells while observing them on the stage of an inverted microscope. (3) Only 2 μl of sample solution is sufficient. (4) The device is very easy to use, as the cuvette, which is connected to the tip of a commercially available auto-pipette, is manipulated by hand. Using our device, we loaded a fluorescent probe of actin filaments, Alexa Fluor 546 phalloidin, into migrating keratocytes. The level of this probe in the cells could be easily adjusted by changing its concentration in the electroporation medium. Samples could be loaded into keratocytes, neutrophil-like HL-60 cells and Dictyostelium cells on a coverslip, and keratocytes on an elastic silicone substratum. The new device should be useful for a wide range of adherent cells and allow electroporation for cells on various types of the substrata.
标记特定分子及其在活细胞中的人为控制是研究细胞内分子动力学的有力技术。为了使用这些技术，需要将分子化合物（以下简称“样品”）加载到细胞中。电穿孔技术用于将不可渗透细胞膜的样品加载到细胞中。在这里，我们开发了一种具有四个特殊特性的新型电穿孔仪。（1）将电脉冲直接施加到贴壁细胞上，而无需将其从基质上移除。（2）可以在倒置显微镜的载物台上观察到贴壁细胞的同时将样品加载到贴壁细胞中。（3）仅需 2 μl 的样品溶液即可。（4）该设备非常易于使用，因为通过手动操作与市售自动移液器连接的试管即可。使用我们的设备，我们将肌动蛋白丝的荧光探针，Alexa Fluor 546 鬼笔环肽加载到迁移的角膜细胞中。通过改变电穿孔介质中探针的浓度，可以轻松调节细胞中探针的水平。可以将样品加载到盖玻片上的角膜细胞、类中性粒细胞 HL-60 细胞和盘基网柄菌细胞以及弹性硅酮基质上的角膜细胞中。这种新型设备应该对各种贴壁细胞都有用，并允许对各种基质上的细胞进行电穿孔。
