A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons.
A fluorescence recovery after photobleaching protocol to measure surface diffusion of DAGLα in primary cultured cortical mouse neurons.
复制标题
在原代培养的小鼠皮层神经元中，光漂白方案后的荧光恢复用于测量DAGLα的表面扩散。
DOI：
10.1016/j.xpro.2021.101118
复制
发表时间：
2022-03-18
期刊：
影响因子：
--
通讯作者：
Penzes P
中科院分区：
文献类型：
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作者：
Yoon S;Penzes P
该协议描述了使用超黄 pHluorin (SEP)-二酰甘油脂肪酶 α (DAGLα) 光漂白 (FRAP) 后的荧光恢复来测量原代培养皮质小鼠神经元树突轴中膜结合 DAGLα 的迁移率。这可以作为分析内源性大麻素介导的突触可塑性的绝佳工具。我们使用该协议来表明 DAGLα 表面动力学在调节树突棘中发挥着不可或缺的作用。我们还详细介绍了如何通过 FRAP 测定测试 HEK293T 细胞中生成的 SEP-DAGLα 的质量。有关此配置文件的使用和执行的完整详细信息，请参阅。通过将 SEP 插入第一个胞外域生成 SEP-DAGLα HEK293T 细胞中 SEP-DAGLα 的表达和功能测试 SEP-DAGLα 的 FRAP 以确定 DAGLα 在树突轴中的扩散 该方案描述了使用超黄 pHluorin (SEP)-二酰基甘油脂肪酶 α (DAGLα) 光漂白 (FRAP) 后的荧光恢复来测量膜结合 DAGLα原代培养的皮质小鼠神经元树突轴的移动性。这可以作为分析内源性大麻素介导的突触可塑性的绝佳工具。我们使用该协议来表明 DAGLα 表面动力学在调节树突棘中发挥着不可或缺的作用。我们还详细介绍了如何通过 FRAP 测定测试 HEK293T 细胞中生成的 SEP-DAGLα 的质量。
This protocol describes using fluorescence recovery after photobleaching (FRAP) of a superecliptic pHluorin (SEP)-diacylglycerol lipase α (DAGLα) to measure membrane-bound DAGLα mobility in dendritic shafts of primary cultured cortical mouse neurons. This could serve as an excellent tool to analyze endocannabinoid-mediated synaptic plasticity. We have used this protocol to show that DAGLα surface dynamics play an integral role in regulating the dendritic spine. We also detail how we test the qualities of generated SEP-DAGLα in HEK293T cells by FRAP assay. For complete details on the use and execution of this profile, please refer to. Generation of SEP-DAGLα by inserting SEP into the first extracellular domain Expression and functional tests of SEP-DAGLα in HEK293T cells FRAP of SEP-DAGLα to determine DAGLα diffusion in dendritic shafts This protocol describes using fluorescence recovery after photobleaching (FRAP) of a superecliptic pHluorin (SEP)-diacylglycerol lipase α (DAGLα) to measure membrane-bound DAGLα mobility in dendritic shafts of primary cultured cortical mouse neurons. This could serve as an excellent tool to analyze endocannabinoid-mediated synaptic plasticity. We have used this protocol to show that DAGLα surface dynamics play an integral role in regulating the dendritic spine. We also detail how we test the qualities of generated SEP-DAGLα in HEK293T cells by FRAP assay.