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Abstract：
Loss of plasma membrane lipid asymmetry contributes to many cellular functions and responses, including apoptosis, blood coagulation, and cell fusion. In this protocol, we describe the use of fluorescently labeled annexin V to detect loss of lipid asymmetry in the plasma membrane of adherent living cells by fluorescence microscopy. The approach provides a simple, sensitive, and reproducible method to detect changes in lipid asymmetry but is limited by low sample throughput. The protocol can also be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. To validate the lipid binding properties of such probes, we additionally describe here the preparation and use of giant unilamellar vesicles as simple model membrane systems that have a size comparable to cells. Key features Monitoring loss of lipid asymmetry in the plasma membrane via confocal microscopy. Protocol can be applied to any type of cell that is adherent in culture, including primary cells. Assay can be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. Giant unilamellar vesicles serve as a tool to validate the lipid binding properties of such probes. Graphical overview Imaging the binding of fluorescent annexin V to adherent mammalian cells and giant vesicles by confocal microscopy. Annexin V labeling is a useful method for detecting a loss of plasma membrane lipid asymmetry in cells (top image, red); DAPI can be used to identify nuclei (top image, blue). Giant vesicles are used as a tool to validate the lipid binding properties of annexin V to anionic lipids (lower image, red).
摘要：
质膜脂质不对称性的丧失有助于许多细胞功能和反应，包括细胞凋亡,血液凝固，和细胞融合。在这个协议中,我们描述了使用荧光标记的膜联蛋白V通过荧光显微镜检测粘附活细胞质膜中脂质不对称的损失。该方法提供了一个简单的,敏感,和可重复的方法来检测脂质不对称的变化，但受到低样品通量的限制。该方案还可以适用于其他荧光标记的脂质结合蛋白或肽探针。为了验证此类探针的脂质结合特性，我们还在这里描述了巨大的单层囊泡的制备和使用，作为简单的模型膜系统，其大小与细胞相当。通过共聚焦显微镜监测质膜中脂质不对称性的丧失。方案可以应用于培养中粘附的任何类型的细胞，包括原代细胞。测定可以适用于其他荧光标记的脂质结合蛋白或肽探针。巨大的单层囊泡用作验证此类探针的脂质结合特性的工具。图形概述通过共聚焦显微镜成像荧光膜联蛋白V与粘附哺乳动物细胞和巨囊泡的结合。膜联蛋白V标记是检测细胞中质膜脂质不对称性丢失的有用方法（上图，红色)；DAPI可用于识别细胞核(顶部图像，蓝色)。巨囊泡被用作验证膜联蛋白V与阴离子脂质的脂质结合特性的工具（下图，红色)。
