Ex vivo Assessment of Mitochondrial Function in Human Peripheral Blood Mononuclear Cells Using XF Analyzer
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Author:
Date:
2021-04-05
[Abstract] Cellular health and function, as we know today, depend on a large extent on mitochondrial function. The essential function of mitochondria is the energy production, more precisely ATP production, via oxidative phosphorylation. Mitochondrial energy production parameters therefore represent important biomarkers. Studies on human cells have mainly been performed on in vitro cell cultures. However, peripheral blood mononuclear cells (PBMCs) are particularly suitable for such examinations. That’s why this protocol describes a method to measure key parameters of mitochondrial function in freshly isolated PBMCs with the latest technology, the XF Analyzer. For this ex vivo approach PBMCs are first isolated out of human anticoagulated blood. Next, they are attached to the surface of special ...
[摘要] [摘要]正如我们今天所知,细胞的健康和功能在很大程度上取决于线粒体的功能。线粒体的基本功能是ENER GY生产,更精确的LY ATP生产,通过氧化磷酸化。因此,线粒体能量产生参数代表重要的生物标记。对人类细胞的研究主要是在体外细胞培养中进行的。然而,外周血单核细胞(PBMC)是特别升ý适于这样的检查。这就是为什么这个协议描述测量与最新的技术,新鲜分离的PBMC线粒体功能的关键参数的方法的XF分析。对于这个离体PBMC首先是从人抗凝血液中分离出来的。接下来,将它们附着到预先涂有Poly-D-Lysine的特殊微孔板的表面上。期间的氧消耗速率(OCR)以及细胞外酸化率(ECAR)的应力试剂寡,羰氰化物随后的测量4 - (三氟甲氧基)苯腙(FCCP),鱼藤酮和抗霉素A被注入。可以从获得的结果中计算出几个线粒体参数。该协议的应用允许分析对人体细胞的各种影响,例如药物或环境因素。
[背景]线粒体在维持正常细胞功能中起关键作用。现在众所周知,它们不仅通过氧化磷酸化产生ATP,而且还参与氨基酸,脂质和核苷酸的代谢,潜水信号转导和氧化还原过程以及质量控制和降解过程,包括线粒体和磷酸化。细胞凋亡(Pfanner等,2019)。然而,线粒体代表正常细胞中ATP合成的主要位点(Akbari等人,2019)。为此,通过多亚基酶复合物I – ...
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Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts
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Author:
Date:
2021-03-20
[Abstract] 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead ...
[摘要] [摘要] 7,8-二氢-8-氧鸟嘌呤(8-oxoG)是由活性氧(ROS)引起的最常见且诱变的氧化DN A损伤之一。由于ROS主要在线粒体的内膜中产生,因此这些细胞器,特别是其中所含的线粒体DNA(mtDNA)受到这种损害的特别影响。消除8-oxoG可能会导致突变,从而导致严重的线粒体功能障碍。为了消除8-oxoG,人体使用了8-氧代鸟嘌呤DNA糖基化酶1(OGG1),它是DNA氧化损伤的主要拮抗剂。但是,先前的研究表明,人类OGG1的活性(h OGG1)随着年龄的增长而减少,导致与年龄相关的8-oxoG积累。更好地了解hOGG1的确切机制可能会导致发现新的靶标,因此对于开发预防性疗法具有重要意义。因此,我们开发了一种实时碱基切除修复测定法,该测定法采用了专门设计的双链报告寡核苷酸来测量分离的线粒体裂解物中hOGG1的活性。这里介绍的该系统与经典测定法不同,在经典测定法中,可以通过实时测量hOGG1活性通过变性丙烯酰胺凝胶进行终点测定。另外,为了确定该双功能酶的每个酶促步骤的活性(N-糖基化酶和AP-裂解酶活性),还可以进行解链曲线分析。使用各种离心步骤从人成纤维细胞中分离线粒体后,将其裂解,然后与专门设计的报告寡核苷酸一起孵育。hOGG1活性的后续测量是在常规实时PCR系统中进行的。
[背景]人体是永久的损害案例。每天约10 ...
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A Transient Transfection-based Cell Adhesion Assay with 293T Cells
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Author:
Date:
2021-01-05
[Abstract] The in vitro cell adhesion assay is a quantitative method for measuring selective cell adhesion to specific proteins. Traditionally, cell adhesion assays employ purified protein immobilized on a solid glass or plastic surface. Here, we describe a transient 293T cell transfection-based cell adhesion assay to study selective cell adhesion of a specific cell type to a protein of interest. In this protocol, 293T cells are transfected with a mammalian expression plasmid containing mSiglec1 cDNA or an empty plasmid as a mock control and are then cultured to form a monolayer. Subsequently, these Siglec1-expressing and mock-transfected 293T cell monolayers are used for cell adhesion assays with GFP-expressing B16F10 cells. The number of GFP+ cancer cells adhering to each 293T monolayer is a ...
[摘要] [摘要]的体外细胞粘附分析是一种用于测量到特定蛋白选择性细胞粘附的定量方法。传统上,细胞粘附测定采用固定在固体玻璃或塑料表面上的纯化蛋白质。在这里,我们描述了基于瞬时293T细胞转染的细胞粘附试验,以研究特定细胞类型对目标蛋白质的选择性细胞粘附。在该协议中,将293T细胞用包含mSiglec1 cDNA的哺乳动物表达质粒或空质粒作为模拟对照转染,然后培养以形成单层。随后,将这些表达Siglec1和模拟转染的293T细胞单层用于表达GFP的B16F10细胞的细胞粘附测定。GFP +的数量 粘附在每个293T单层上的癌细胞是一种定量手段,用于比较癌细胞与Siglec1的选择性粘附性。该方法消除了表达和纯化目的蛋白以进行体外细胞粘附测定的需要,并且可以容易地用难以纯化的蛋白进行操作,同时保持其天然的原位结构。
关键词:细胞粘附试验,细胞粘附,癌细胞粘附试验,293T,瞬时转染,Siglec1,F荧光显微镜
[背景]细胞-细胞相互作用对于生物学过程,例如组织发育,再生,和临界形态发生,以及免疫应答和癌症转移(Gumbiner,1996 ...
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