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Sodium pyruvate

丙酮酸钠

公司名称: Sigma-Aldrich
产品编号: P2256
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Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures
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Date:
2017-06-05
[Abstract]  The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell cultures serve as a practicable tool for quantification of cellular injury (Koh and Choi, 1987; Bruer et al., 1997). Lactate dehydrogenase (LDH) is such a ubiquitously expressed cytosolic enzyme, which is very stable due to a ... [摘要]  神经生物学领域中许多急性或慢性退行性疾病的体外模型的目的是评估神经元细胞的存活或损伤。细胞损伤与外细胞膜完整性的丧失和细胞质细胞蛋白的泄漏有关。因此,细胞培养上清液中细胞质酶的活性测定作为细胞损伤定量的实用工具(Koh和Choi,1987; Bruer等,1997)。乳酸脱氢酶(LDH)是一种无处不在表达的细胞溶质酶,由于蛋白质的半衰期很长,其稳定性很高(Hsieh和Blumenthal,1956; Koh和Cotman,1992; Koh等人)。 ,1995)。

背景 LDH在可逆的生物化学反应中催化丙酮酸和还原的烟酰胺偶氮二核苷酸(NADH)的乳酸盐和烟酰胺氨基茚三核苷酸(NAD +)的形成。 NADH在340nm的波长上具有吸收。这种动力学LDH活性测定的基础是由NADH降低引起的特定波长处的光密度降低。使用具有已知LDH活性的标准酶溶液计算上清液中LDH的量。不同的细胞密度或代谢活化率可能是混杂的;因此推荐LDH活性的正常化。这是通过评估外部细胞膜裂解后不抑制LDH活性的LDH活性(用0.5%Triton-X“完全杀死”)来实现的。最后,通过完全杀死的LDH活性的绝对LDH活性百分比表示细胞培养物中损伤或死细胞的发生率。

A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
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Date:
2017-06-05
[Abstract]  The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 ... [摘要]  聚类规则间隔短回文重复(CRISPR)/ CRISPR相关蛋白9(Cas9)系统是使用最广泛的基因组编辑工具。一个常见的CRISPR / Cas9系统由两个组成部分组成:单导RNA(sgRNA)和Cas9。在特定靶序列引入双链断裂(DSB)需要两种成分。该系统的一个缺点是实验室中sgRNA的生产是费力的,因为它需要在体外​​转录反应和sgRNA纯化之间克隆sgRNA序列。通过sgRNA靶向Cas9活性的替代方案是用两种小RNA:CRISPR RNA(crRNA)和反式激活性crRNA(tracrRNA)进行靶向。这两种小RNA可以化学合成,这使得与sgRNA相比,这些RNA的产生不那么困难。 CRISPR / Cas9系统的另一个缺点是已经报告了脱靶效应。然而,已经开发了改进形式的Cas9以最小化离靶效应。例如,仅当两个引导RNA在规定的距离内结合相对的链时,切口酶型Cas9(nCas9)和FokI结构域融合的催化无活性的Cas9(FokI-dCas9; fCas9)才诱导DSB。在本协议中,我们描述了使用结合crRNA,tracrRNA和Cas9修饰形式的CRISPR / Cas9系统来生产突变小鼠的实验系统。该方法不仅有利于制备用于基因组编辑系统的试剂,而且可以降低脱靶效应的风险。

背景 ...

Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime
Author:
Date:
2017-01-20
[Abstract]  NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be ... [摘要]  NADH和NADPH分别是分解代谢和合成代谢过程的氧化还原辅因子。此外,NADPH在细胞抗氧化防御中起着重要作用。在活细胞和组织中,其光谱相同的自发荧光(称为NAD(P)H)的强度可用于探测线粒体氧化还原状态,而其不同的酶结合特征可用于将其相对贡献与总共分离使用荧光寿命成像显微镜(FLIM)的NAD(P)H强度。这些方案允许在细胞类型和改变的生理和病理状态之间检测代谢的差异。

背景 氧化还原辅因子烟酰胺腺嘌呤二核苷酸(NADH)及其磷酸化对应物NADPH的还原形式本质上是荧光的,两者都吸收波长为340(±30)nm并在460(±50)nm处发射的光(Patterson等人。,2000)。这些光谱特征在氧化成NAD(上标+)或NADP(superson),(2007))时损失。单独的NAD和NADP池的氧化还原平衡决定了对比的代谢过程(Ying,2008),如图1所示。NAD作为电子受体,用于通过三羧酸氧化线粒体中的糖,脂质和氨基酸底物(TCA)循环,并作为内线粒体膜(IMM)上的电子传递链(ETC)的电子供体,促使将质子泵送到膜间隙中,作为合成三磷酸腺苷(ATP)的电源,通过F 1 F 0 O 3 ATP合成酶(Osellame等人,2012)。因此,线粒体中NADH与NAD + 的平衡反映了TCA循环与ETC活性的平衡。 ...

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