Peng H B, Jaffe L F
J Cell Biol. 1976 Nov;71(2):674-80. doi: 10.1083/jcb.71.2.674.
A simple and selective method for freeze-fracturing spherical cells is described. The cells are loaded into the holes of a thin nickel screen. A metal hat is applied to the cell monolayer and the whole assembly, hat-cells-screen, is frozen and then fractured by ripping the hat off. The fractured face on the screen is replicated. By varying the size of the screen holes and by applying the hat to either side of the screen, this method can selectively expose the E face (or the outer half of plasma membrane), the P face (or the inner half of the plasma membrane), or the cytoplasm of the cells. It also provides a means to produce fractures at a preselected area on the cell, if the cells can be loaded onto the screen in an oriented fashion.
描述了一种用于冷冻断裂球形细胞的简单且选择性的方法。将细胞加载到薄镍筛的孔中。将一个金属帽应用于细胞单层,然后将整个组件(帽 - 细胞 - 筛)冷冻,接着通过撕下帽子使其断裂。筛上的断裂面进行复制。通过改变筛孔的大小以及将帽子应用于筛的任一侧,该方法可以选择性地暴露E面(或质膜的外半部分)、P面(或质膜的内半部分)或细胞的细胞质。如果细胞能够以定向方式加载到筛上,它还提供了一种在细胞上的预选区域产生断裂的方法。