Short-term high-resolution imaging of developing hippocampal neurons in culture.
Short-term high-resolution imaging of developing hippocampal neurons in culture.
复制标题
DOI：
10.1101/pdb.prot068247
复制
发表时间：
2012-03-01
影响因子：
--
通讯作者：
Banker, Gary
中科院分区：
文献类型：
--
作者：
Kaech, Stefanie;Huang, Chun-Fang;Banker, Gary
啮齿动物海马体的分离细胞培养物已成为研究神经发育许多方面的标准模型。培养物是非常同质的，并且通过转染相对容易表达绿色荧光蛋白（GFP）标记的蛋白质。因为培养物基本上是二维的，所以不需要在多个焦平面处获取图像。为了以高分辨率捕获快速的亚细胞事件，如本文所述，必须最大化弱信号并减少背景荧光。因此，这些方法在几个方面不同于用于延时成像的方法。脂质体介导的转染产生比用核转染装置转染更高水平的表达。通常使用旋转圆盘共聚焦显微镜收集图像，这提高了信噪比。此外，我们使用了一种成像介质，旨在最大限度地减少背景荧光，而不是提高长期细胞存活率。选择处于适当发育阶段的培养物也很重要。在我们的手中，基于脂质体的转染在接种后3至10天的细胞上效果最好。基于GFP的荧光可以在向细胞中加入DNA/脂质复合物后4小时观察到，但表达通常在接下来的12小时内增加，并保持稳定数天。DNA与脂质的比例是至关重要的;为了降低标记构建体的表达水平，我们使用表达载体和空质粒的组合，保持DNA量恒定。包括一个例子来说明基于微管的膜蛋白的囊泡运输的成像。
Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Because the cultures are essentially two dimensional, there is no need to acquire images at multiple focal planes. For capturing rapid subcellular events at high resolution, as described here, one must maximize weak signals and reduce background fluorescence. Thus, these methods differ in several respects from those used for time-lapse imaging. Lipofectamine-mediated transfection yields a higher level of expression than does transfection with a nucleofection device. Images are usually collected with a spinning-disk confocal microscope, which improves the signal-to-noise ratio. In addition, we use an imaging medium designed to minimize background fluorescence rather than to enhance long-term cell survival. It is also important to select cultures at an appropriate stage of development. In our hands, lipofectamine-based transfection works best on cells between 3 and 10 d after plating. GFP-based fluorescence can be observed as early as 4 h after adding the DNA/lipid complexes to the cells, but expression usually increases over the next 12 h and remains steady for days. The ratio of DNA to lipid is critical; to lower expression levels of the tagged construct, we use a combination of expression vector and empty plasmid, keeping the DNA amount constant. An example is included to illustrate the imaging of the microtubule-based vesicular transport of membrane proteins.