| Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime
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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 detected between cell types and altered physiological and pathological states.
[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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| PhagoKinetic Track Assay: Imaging and Analysis of Single Cell Migration
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Author:
Date:
2016-01-05
[Abstract] Cell migration is a highly complex and dynamic biological process, essential in several physiological phenomena and pathologies including cancer dissemination and metastasis formation. Thus understanding single cell migration is highly relevant and requires a suitable image-based assay. Depending on the speed of the moving cells, one may require fast time-lapse microscopy, which is not always suitable for high-throughput screening. To overcome this, a quantitative and fixed single cell migration assay was developed based on the PhagoKinetic Tracks (PKT) procedure. Briefly, cells are seeded on top of a monolayer of carboxylated latex beads, and as cells migrate, they phagocytose these beads and leave behind a migratory track. These bead-free migratory tracks can be visualized using a ...
[摘要] 细胞迁移是高度复杂和动态的生物过程，在几种生理现象和病理包括癌症传播和转移形成中是必需的。因此，理解单细胞迁移是高度相关的，并需要合适的基于图像的测定。根据移动细胞的速度，可能需要快速时间延迟显微术，这不总是适合于高通量筛选。为了克服这一点，基于Phago运动轨迹（PKT）程序开发了定量和固定的单细胞迁移测定。简言之，将细胞接种在单层羧化的乳胶珠的顶部，并且当细胞迁移时，它们吞噬这些珠并留下迁移轨迹。这些无珠移动轨迹可以使用标准明场显微镜可视化，并分析单细胞迁移的多参数定量评估（Naffar-Abu-Amara等人，2008）。
在这里，我们描述了PKT测定的详细和优化的方案，适用于RNAi和药物筛选（van Roosmalen等人，2015）。这个协议允许用户在单细胞水平研究迁移行为，没有快速和活成像显微镜。
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| Scratch Wound Healing Assay
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Author:
Date:
2012-03-05
[Abstract] The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer is created by scratching, and the “healing” of this gap by cell migration and growth towards the center of the gap is monitored and often quantitated. Factors that alter the motility and/or growth of the cells can lead to increased or decreased rate of “healing” of the gap (Lampugnani, 1999). This assay is simple, inexpensive, and experimental conditions can be easily adjusted for different purposes. The assay can also be used for a high-throughput screen platform if an automated system ...
[摘要] 划伤伤口愈合测定法已经广泛适应和修改以研究多种实验条件例如基因敲低或化学暴露对哺乳动物细胞迁移和增殖的影响。 在典型的伤口创伤愈合测定中，通过刮擦产生细胞单层中的"伤口间隙"，并且监测并常常定量通过细胞迁移和朝向间隙中心的生长的该间隙的"愈合"。 改变细胞运动性和/或生长的因素可导致间隙"愈合"的增加或减少的速率（Lampugnani，1999）。 该测定简单，便宜，并且可以容易地针对不同目的调整实验条件。 如果使用自动化系统，该测定也可以用于高通量筛选平台（Yarrow和Perlman，2004）。
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