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A Sensitive and Quantitative mKeima Assay for Mitophagy via FACS

机译:通过FACS的乳化物敏感和定量的Mkea测定法

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Mitophagy is a selective autophagy process that specifically removes damaged mitochondria via general autophagy. The two major recessive Parkinson's disease genes PINK1 and Parkin play essential roles in mitophagy initiation, increasing the interest in mitophagy in both basic and translational research over the past 10 years. Initially, mitophagy was measured by the loss of mitochondria either through confocal imaging or immunoblot of mitochondrial proteins such as Tom20 or COXII. Confocal imaging ofmitochondria DNA loss via anti‐DNA staining is another option. All of these methods, which take considerable effort and labor, are not sensitive enough to detect early stages of mitophagy with unambiguous objectivity. The mKeima assay can be used for both confocal imaging and FACS analysis to provide a thorough picture of mitophagy with a wide dynamic range. The Keima fluorophore has bimodal excitation under neutral and acidic pH conditions. Thus, when Keima is targeted to mitochondria it can accurately reveal the formation of mitolysosomes. Here the author briefly describes the origin and history of mKeima and how it is adapted to measure mitophagy. The author presents detailed protocols for making stable cell lines for optimized mitophagy detection and discuss many parameters that might affect the assay. A troubleshooting section is also provided to discuss possible pitfalls to improve reproducibility and sensitivity of the assay.
机译:mitophagy是一种选择性自噬过程,可通过一般自噬地清除受损的线粒体。两种主要的隐性帕金森病基因Pink1和Parkin在过去10年中增加了在基本和翻译研究中对肠系的兴趣增加。最初,通过通过共聚焦成像或线粒体蛋白质如汤姆20或COXII来损失线粒体的丧失来测量MITOCHAGY。通过抗DNA染色的粒细胞DNA损失的共聚焦成像是另一种选择。所有这些方法都采取了相当大的努力和劳动力,不足以检测与明确的客观性的影响的早期阶段。 Mkeima测定可用于共聚焦成像和FACS分析,以提供具有宽动态范围的彻底图像。 Keima荧光团在中性和酸性pH条件下具有双峰激发。因此,当Keima靶向线粒体时,它可以准确地揭示MitolysoSomes的形成。在这里,作者简要介绍了Mkeima的起源和历史以及如何适应测量肠果。作者介绍了制备稳定细胞系的详细方案,用于优化的细胞系检测,并讨论可能影响测定的许多参数。还提供了故障排除部分以讨论可能的缺陷,以提高测定的再现性和敏感性。

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