Metal sandwich method to quick-freeze monolayer cultured cells for freeze-fracture
Metal sandwich method to quick-freeze monolayer cultured cells for freeze-fracture
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DOI：
10.1177/002215549704500411
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发表时间：
1997-04-01
影响因子：
3.2
通讯作者：
Fujimoto, K
中科院分区：
文献类型：
--
作者：
Fujimoto, T;Fujimoto, K
我们描述了一种简单的快速冷冻方法，从单层培养的细胞中获得大的质膜断裂平面。细胞生长在薄金箔上，倒置在薄铜箔上的薄明胶层上，并通过在液氮中预冷的两个镀金铜块之间的快速按压来冷冻。将冷冻的细胞夹层以金面朝上安装在冷冻断裂装置的冷台上，并通过用冷断裂刀分离夹层来断裂。当这种技术应用于融合的单层细胞，大副本的E-面对的上质膜和P-面对的下质膜。本发明的金属夹层方法简单，不需要任何昂贵的设备，并且为随后的组织化学操作提供了质膜的大断裂平面。
We describe a simple quick-freezing method to obtain a large fractured plane of the plasma membrane from monolayer cultured cells, Cells were grown on thin gold foil, inverted on a thin layer of gelatin on thin copper foil, and frozen by a quick press between two gold-plated copper blocks precooled in liquid nitrogen. The frozen cell sandwich was mounted on the cold stage of a freeze-fracture device with the gold side up and was fractured by separating the sandwich with a cold fracture knife. When this technique was applied to confluent monolayer cells, large replicas of the E-face of the upper plasma membrane and the P-face of the lower plasma membrane were obtained. The present metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation.