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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Neutrophil Extracellular Trap Killing Assay of Candida albicans
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Author:
Date:
2020-08-20
[Abstract] Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized C. albicans; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized C. albicans. Neutrophil killing of opsonized C. albicans requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of C. albicans requires ...
[摘要] [摘要 ] 丰人病原体念珠菌白色念珠菌是顶级领先的原因之一全球总卫生保健相关血流感染。中性粒细胞是清除白色念珠菌感染的主要效应细胞。我们的研究表明,小鼠中性粒细胞利用两种独立的机制杀死白念珠菌:一种是CR3下游NADPH氧化酶依赖性机制,它可以杀死调理过的白色念珠菌。另一个是dectin-2介导的NADPH氧化酶非依赖性中性粒细胞胞外捕获物(NET),它杀死未调理的白色念珠菌。中性粒细胞杀死调理过的白色念珠菌 需要吞噬生物体并产生活性氧(ROS)。大多数现有的协议的测定中性粒细胞杀死白色念珠菌需要使嗜中性粒细胞吞噬生物体后洗涤步骤。根据定义,NET在细胞外杀死生物。因此,重要的是要跳过洗涤步骤,并向孔中添加最佳比例的嗜中性白细胞和白色念珠菌。为了证明NET的作用,有必要比较用微球菌核酸酶(MNase )(一种消化NET的酶)处理的嗜中性粒细胞的杀伤能力与用热灭活的MNase 处理的嗜中性粒细胞的杀伤能力。MNase 还用于释放与NET绑定的真菌元素以进行计数。该协议可用于测定其他生物膜形成生物的NET杀灭。
[背景 ] ...
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Generation of Functional Mouse Hippocampal Neurons
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Author:
Date:
2020-08-05
[Abstract] Primary culture of mouse hippocampal neurons is a very useful in vitro model for studying neuronal development, axonal and dendritic morphology, synaptic functions, and many other neuronal features. Here we describe a step-by-step process of generating primary neurons from mouse embryonic hippocampi (E17.5/E18.5). Hippocampal neurons generated with this protocol can be plated in different tissue culture dishes according to different experimental aims and can produce a reliable source of pure and differentiated neurons in less than one week. This protocol covers all the steps necessary for the preparation, culture and characterization of the neuronal culture, including the illustration of dissection instruments, surgical procedure for embryos’ isolation, culturing conditions and ...
[摘要] [摘要] 原代培养小鼠海马神经元是一种非常有用的体外模型用于研究神经元的发育,轴突和树突的形态,突触功能,以及许多其他神经元的特征。这里我们描述了从小鼠胚胎海马(E17.5/E18.5)产生初级神经元的一步一步的过程。根据不同的实验目的,用该方法产生的海马神经元可以在不同的组织培养皿中进行培养,并能在不到一周的时间内产生一个可靠的来源。该方案涵盖了神经元培养物的制备、培养和鉴定的所有必要步骤,包括解剖器械的说明、胚胎分离的手术程序、培养条件以及培养物纯度和分化的评估。通过分析培养6天时的钙显像动力学来评估神经元的活性。
[背景] 海马体是一个非常典型的大脑结构,对重要的大脑功能如记忆、空间导航、情绪记忆和学习至关重要。从解剖学上讲,小鼠海马体有一个清晰的C形结构,很容易定位和分离。在细胞水平上,它主要由锥体细胞组成,与其他脑区相比,中间神经元和胶质细胞较少(Kaech和Banker,2006)。因此,海马体是从野生型或基因工程小鼠模型中产生高纯度原代神经元培养物的理想区域,可用于疾病建模或研究神经元功能的多个方面,如突触传递和电生理特性、对神经毒性的敏感性,分化与衰老(;;;;)。Busche,2018Koyama和Ikegaya,2018Molnar,2011Wu等人,2019Rush等人,2020年
已经制定了许多协议,通过与神经胶质喂食器共同培养神经元来产生皮层和海马神经元(Kaech和Banker,2006),描述了用水凝胶微纤维封装的星形胶质细胞的三维神经元培养系统(Kim等人,2020年),长期向培养基中补充生长因子神经元培养(Ray ...
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