High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Jonathan G.T. Lam; Chi Song; Stephanie Seveau

doi:10.3791/58351

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

哺乳动物细胞血浆膜检测效率的高通量测量

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10:07 min

January 7, 2019

DOI: 10.3791/58351-v

Jonathan G.T. Lam^1,2,3, Chi Song⁴, Stephanie Seveau^1,2,3

¹Department of Microbial Infection and Immunity,The Ohio State University, ²Department of Microbiology,The Ohio State University, ³Infectious Diseases Institute,The Ohio State University, ⁴Division of Biostatistics, College of Public Health,The Ohio State University

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在这里, 我们描述了一种高通量荧光检测, 通过在活细胞中的荧光和成像分析测量质膜的重接受效率。该检测方法可用于筛选调节哺乳动物细胞质膜重密封的药物或靶向基因。

该检测是为了解决等离子膜生物学领域关键问题，并确定受损细胞如何有效地重新密封其等离子膜。该技术可用于测量高通量容量的等离子膜再密封效率，并确定调节等离子膜再密封的特定蛋白质和相应通路。首先将20毫升的2.5倍10添加到每毫升生长介质悬浮液的第五个 HeLa 细胞，加入无菌移液器盆，并使用 10 毫升血清移液器将细胞彻底混合。

接下来，使用多通道微移子在96孔平坦、清澈、黑色聚苯乙烯组织培养的培养板中，每孔中播种100微升细胞。请记住，同质细胞分布是成功实验的关键，因为所有实验条件之间的比较都需要等效的细胞计数。在37摄氏度和5%CO2的加湿细胞培养箱中24小时后，将板读卡器预热至37摄氏度，将光学配置设置为单色器，读取模式设置为荧光，读取类型设置为动力学。

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