Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair
Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair
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DOI：
10.3791/51106
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发表时间：
2014-03-01
影响因子：
1.2
通讯作者：
Jaiswal, Jyoti K.
中科院分区：
文献类型：
--
作者：
Defour, Aurelia;Sreetama, S. C.;Jaiswal, Jyoti K.
受伤细胞的愈合能力是一个基本的细胞过程，但涉及愈合受伤细胞的细胞和分子机制知之甚少。本文描述了监测局部损伤后培养细胞愈合的能力和动力学的测定。第一个方案描述了一种基于终点的方法，以同时评估数百个细胞的细胞膜修复能力。第二个协议描述了一个真实的时间成像方法，以监测动力学的细胞膜修复后，在个别细胞局部损伤的脉冲激光。由于愈合受损细胞涉及将特定蛋白质和亚细胞区室运输到损伤部位，第三项议定书描述了使用上述基于终点的方法来评估此类贩运事件（溶酶体胞吐）最后一个方案描述了使用脉冲激光损伤与TIRF显微镜一起监测损伤细胞中单个亚细胞区室的动态，高空间和时间分辨率。虽然这里的方案描述了使用这些方法来研究培养的肌细胞中细胞膜修复和溶酶体胞吐之间的联系，但它们可以同样应用于任何其他贴壁培养的细胞和选择的亚细胞区室。
The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.