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2 ml and 1.5 ml Eppendorf tubes

Company: Hounisen
Catalog#: 72.706
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Ex vivo Assessment of Mitochondrial Function in Human Peripheral Blood Mononuclear Cells Using XF Analyzer
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

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[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 – ...

Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE)
Author:
Date:
2021-03-05
[Abstract]  

Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The

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[摘要]  [摘要]非洲锥虫和其他原生动物生物线粒体内的基因表达依赖于核苷酸特异性的RNA编辑反应。在该过程中,仅将尿苷(U)-核苷酸位点特异性插入序列不足的初级转录物中,并从中缺失,以将其转化为可翻译的mRNA。该反应由0.8 MDa的多蛋白复合物催化,该复合物被称为编辑体。在这里我们描述了一种改进的体外试验,以定量探索Editosome的催化活性。该测定使用合成的,荧光团衍生的寡核糖核苷酸 作为编辑底物,可通过耦合到激光诱导荧光(LIF)检测系统的毛细管电泳(CE)自动分离反应产物。该测定法功能强大,只需要纳克级的材料,并且通过使用多毛细管CE / LIF仪器,可以高度平行的方式进行测定。进一步的改进包括使用硫代磷酸酯修饰的,因此具有RNase耐性的底物RNA,以及用于同时监测U插入和U缺失反应的多重型荧光团标记策略。该测定方法对于研究酶体的机理和酶学是有用的。ħ H但是,它也可以在高通量执行以筛选RNA编辑特异性抑制剂。


图形摘要:


基于荧光的体外U插入/ U缺失RNA编辑(FIDE)分析的特征




[背景]中的RNA编辑反应动质体原生动物如非洲锥虫和利什曼原虫表示一个信使的最显着的转录后修饰(米)的RNA(综述Göringer ...

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