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Trypsin-EDTA (0.05%)

胰蛋白酶-EDTA(0.05%)

Company: Thermo Fisher Scientific
Catalog#: 25300062
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Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells
Author:
Date:
2018-08-20
[Abstract]  Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 μm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer’s group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in ... [摘要]  纤连蛋白(FN)是一种细胞外基质蛋白,由许多细胞类型分泌,主要与细胞表面受体整合素α5β1结合。整合素α5β1结合启动FN逐步组装成原纤维,这一过程称为原纤维形成。我们和其他几个人已经证明了原纤维形成对细胞迁移和转移的关键作用。虽然免疫染色和显微镜方法有助于可视化FN掺入原纤维,每个原纤维的长度至少为3μm,但是第一项研究开发了一种生物化学分离FN以量化原纤维并入FN的方法,由Jean Schwarzbauer小组于1996年出版。我们的方案改编自原始出版物,并已在多种细胞类型上进行测试,包括如此处所示的MCF10A乳腺上皮细胞和Caki-1肾癌上皮细胞。使用两种洗涤剂提取物,将细胞FN分离成不溶于洗涤剂或掺入原纤维的FN和可溶性FN或未掺入的级分。为了确定原纤维形成是否利用FN的再循环池,我们使用了生物素标记的FN(FN-生物素)再循环测定,其已经从先前的研究中修改。使用再循环测定和脱氧胆酸盐分离方法的组合,可以定量地证明在不同实验条件下细胞中原纤维形成的程度,并确定原纤维形成的FN来源

【背景】 纤连蛋白(FN)是普遍产生的细胞外基质(ECM)组分(Uitto et al。,1989; Mao和Schwarzbauer,2005)。纤连蛋白库是转录产生的,可以通过几种生长因子如TGF-β1增加(Yokoi et al。,2002; Mimura ...

FACS-based Glucose Uptake Assay of Mouse Embryonic Fibroblasts and Breast Cancer Cells Using 2-NBDG Probe
Author:
Date:
2018-04-20
[Abstract]  This is a flow cytometry-based protocol to measure glucose uptake of mouse embryonic fibroblasts (MEFs) and breast cancer cells in vitro. The method is a slightly modified and updated version as previously described (Dong et al., 2017). Briefly, the target cells are incubated with the fluorescently tagged 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) for 2 h or 30 min, and the efficiency of glucose uptake is examined using a flow cytometer. This method can be adapted to measure a variety of adipocytes, immune cells, MEFs and cancer cells. [摘要]  这是一种基于流式细胞术的方法,用于在体外测量小鼠胚胎成纤维细胞(MEFs)和乳腺癌细胞的葡萄糖摄取。 该方法是稍微修改和更新的版本(Dong等人,2017)。 简言之,将靶细胞与荧光标记的2-(N-(7-硝基苯-2-基-1,3-二唑-4-基)氨基)-2-脱氧葡萄糖(2-NBDG)一起温育2小时或 30分钟,并使用流式细胞仪检查葡萄糖摄取的效率。 该方法可适用于测量各种脂肪细胞,免疫细胞,MEF和癌细胞。

【背景】葡萄糖是细胞的主要能量来源。葡萄糖转运蛋白家族(GLUT)负责跨葡萄糖转运葡萄糖(Kohn等人,1996)。葡萄糖摄取的变化可以反映细胞代谢的变化。例如,肿瘤细胞通常使用葡萄糖进行有氧糖酵解以支持其快速增殖。通常,与正常细胞相比,肿瘤细胞具有增加的葡萄糖摄取速率(Vander Heiden等人,2009)。 2-脱氧葡萄糖(2DG)是一种葡萄糖类似物,它以2-脱氧葡萄糖-6-磷酸(2DG6P)的形式积累在细胞中。 2DG6P长期以来一直是测量葡萄糖摄取的黄金标准(Yamamoto et al。,2011)。尽管放射性标记的2DG6P的测量是敏感的,但许多研究人员避免了这种方法,因为放射性物质的处理和处置需要特殊的程序。

另一种不可代谢的葡萄糖类似物是荧光标记的2-(N-(7-硝基苯-2-氧杂-1,3-二唑-4-基)氨基)-2-脱氧葡萄糖(2-NBDG)。该分子通过葡萄糖转运蛋白积聚在活细胞中,不会进入糖酵解途径。 ...

Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor
Author:
Date:
2018-01-20
[Abstract]  Reactive oxygen species (ROS) are not only known for their toxic effects on cells, but they also play an important role as second messengers. As such, they control a variety of cellular functions such as proliferation, metabolism, differentiation and apoptosis. Thus, ROS are involved in the regulation of multiple physiological and pathophysiological processes. It is now apparent that there are transient and local changes in ROS in the cell; in so-called ‘microdomains’ or in specific cellular compartments, which affect signaling events. These ROS hotspots need to be studied in more depth to understand their function and regulation. Therefore, it is necessary to identify and quantify redox signals in single cells with high spatial and temporal resolution. Genetically encoded ... [摘要]  活性氧(ROS)不仅以其对细胞的毒性作用而闻名,而且作为第二信使也起着重要的作用。如此,它们控制多种细胞功能,例如增殖,代谢,分化和凋亡。因此,ROS参与多种生理和病理生理过程的调节。现在很明显,细胞内存在ROS的短暂和局部变化;在所谓的“微域”或特定的细胞区室中,其影响信号传导事件。这些ROS热点需要更深入的研究,以了解其功能和规定。因此,有必要以高空间和时间分辨率在单个细胞中识别和量化氧化还原信号。遗传编码的基于荧光的蛋白质传感器提供了检测氧化还原信号传导过程的必要工具。这些传感器的一个很大的优势是可以针对他们。线粒体是能量代谢所必需的,并且是哺乳动物细胞中ROS的主要来源之一。因此,对这些细胞器中氧化还原电位和ROS产生的评估是非常有意义的。在此,我们提供了一个使用H 2 O 2特异性比例传感器mitoHyPer在贴壁哺乳动物细胞中。【背景】ROS是通过线粒体呼吸的副产物,通过电子从电子传递链泄漏而产生的。这些ROS被认为是有毒的,并导致脂质,蛋白质的氧化,并导致线粒体DNA损伤(Ralph et al。,2010; Bogeski等人,2016; Bogeski ,2014; Monika et al。,2015)。虽然线粒体作为新陈代谢,生物能量学和细胞死亡的枢纽,线粒体ROS作为调节多种细胞功能的第二信使的新兴作用也日益被接受(Chandel,2015; ...

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