Lang Thorsten
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Methods Mol Biol. 2008;440:51-9. doi: 10.1007/978-1-59745-178-9_4.
This cell-free assay for exocytosis is particularly useful when spatial information about exocytotic sites and biochemical access to the plasma membrane within less than a minute is required. It is based on the study of plasma membrane lawns from secretory cells exhibiting secretory granules filled with neuropeptide Y-green fluorescent protein (NPY-GFP). The sample is prepared by subjecting NPY-GFP-expressing cells to a brief ultrasound pulse, leaving behind a basal, flat plasma membrane with fluorescent attached secretory organelles. These sheets can then be incubated in defined solutions with the benefit that complete solution changes can be achieved in less than 1 min. Individual secretory granules are monitored in the docked state and during exocytosis by video microscopy.
当需要有关胞吐位点的空间信息以及在不到一分钟的时间内对质膜进行生化分析时，这种无细胞胞吐分析特别有用。它基于对分泌细胞的质膜片层的研究，这些细胞呈现出充满神经肽Y-绿色荧光蛋白（NPY-GFP）的分泌颗粒。通过对表达NPY-GFP的细胞施加短暂的超声脉冲来制备样品，留下带有荧光附着分泌细胞器的基底扁平质膜。然后可以将这些片层在特定溶液中孵育，好处是可以在不到1分钟的时间内实现完全的溶液更换。通过视频显微镜监测单个分泌颗粒在停靠状态和胞吐过程中的情况。
