Immunofluorescence microscopy-based assessment of cytosolic DNA accumulation in mammalian cells.
Immunofluorescence microscopy-based assessment of cytosolic DNA accumulation in mammalian cells.
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DOI：
10.1016/j.xpro.2021.100488
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发表时间：
2021-06-18
期刊：
影响因子：
--
通讯作者：
Galluzzi L
中科院分区：
文献类型：
--
作者：
Sato A;Buque A;Yamazaki T;Bloy N;Petroni G;Galluzzi L
在这里，我们描述了一种免疫荧光（IF）显微镜为基础的方法，以量化在培养的真核细胞的选择性和特定的质膜透化后的胞质双链DNA分子。该技术与宽视野显微镜兼容，并结合自动图像分析用于中高通量应用，以及高分辨率共聚焦显微镜用于亚细胞评估和共定位研究。除了能够实现单细胞和亚细胞分辨率之外，这种方法还规避了与基于亚细胞分级分离的替代方法相关的大多数限制。有关本方案的完整使用和执行，请参阅。高度受控的透化作用能够特异性检测胞质dsDNA与共染色兼容，用于细胞器标记物的共检测适用于共聚焦显微镜，用于高分辨率空间评估适用于宽视野显微镜和自动图像分析，用于定量在这里，我们描述了一种基于免疫荧光（IF）显微镜的方法来定量胞质双-DNA分子在培养的真核细胞中的选择性和特异性质膜透化。该技术与宽视野显微镜兼容，结合自动图像分析用于中高通量应用，以及高分辨率共聚焦显微镜用于亚细胞评估和共定位研究。除了能够实现单细胞和亚细胞分辨率之外，这种方法还规避了与基于亚细胞分级分离的替代方法相关的大多数限制。
Here, we describe an immunofluorescence (IF) microscopy-based approach to quantify cytosolic double-stranded DNA molecules in cultured eukaryotic cells upon the selective and specific permeabilization of plasma membranes. This technique is compatible with widefield microscopy coupled with automated image analysis for mid- to high-throughput applications and high-resolution confocal microscopy for subcellular assessments and co-localization studies. In addition to enabling single-cell and subcellular resolution, this approach circumvents most constraints associated with alternative approaches based on subcellular fractionation. For complete use and execution of this protocol, please refer to. Highly controlled permeabilization enables specific detection of cytosolic dsDNA Compatible with co-staining for the co-detection of organellar markers Amenable to confocal microscopy for high-resolution spatial assessments Amenable to widefield microscopy and automated image analysis for quantitation Here, we describe an immunofluorescence (IF) microscopy-based approach to quantify cytosolic double-stranded DNA molecules in cultured eukaryotic cells upon the selective and specific permeabilization of plasma membranes. This technique is compatible with widefield microscopy coupled with automated image analysis for mid- to high-throughput applications, and high-resolution confocal microscopy for subcellular assessments and co-localization studies. In addition to enabling single-cell and subcellular resolution, this approach circumvents most constraints associated with alternative approaches based on subcellular fractionation.
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