| Qualitative in vivo Bioluminescence Imaging
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Author:
Date:
2018-09-20
[Abstract] Bioluminescence imaging (BLI) technology is an advanced method of carrying out molecular imaging on live laboratory animals in vivo. This powerful technique is widely-used in studying a variety of biological processes, and it has been an ideal tool in exploring tumor growth and metastatic spread in real-time. This technique ensures the optimal use of laboratory animal resources, particularly the ethical principle of reduction in animal use, given its non-invasive nature, ensuring that ongoing biological processes can be studied over time in the same animal, without the need to euthanize groups of mice at specific time points. In this protocol, the luciferase imaging technique was developed to study the effect of co-inoculating pericytes (contractile, αSMA+ mesenchymal ...
[摘要] 生物发光成像（BLI）技术是一种在体内实验室动物上进行分子成像的先进方法。 这种强大的技术广泛应用于研究各种生物过程，是实时探索肿瘤生长和转移扩散的理想工具。 该技术确保实验室动物资源的最佳利用，特别是减少动物使用的伦理原则，考虑到其非侵入性，确保可以在同一动物中随时间研究正在进行的生物过程，而无需安乐死 小鼠在特定的时间点。 在该方案中，开发了荧光素酶成像技术以研究共同接种周细胞（收缩性，αSMA + 间充质干细胞样细胞，位于微血管内的细胞）对生长和转移性扩散的影响。 卵巢癌使用侵袭性卵巢癌细胞系-OVCAR-5-作为例子。
【背景】生物发光成像（BLI）的原理是基于相对简单的生化过程的发光特性，即，荧光素酶介导的分子底物荧光素氧化产生光。在癌症研究中，BLI是一种流行的工具（Contag et ...
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| Ubiquitination Assay for Mammalian Cells
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Author:
Date:
2016-07-20
[Abstract] Ubiquitin is an 8.5 kDa protein that can be activated and conjugated by ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, respectively. Ubiquitin E3 ligases then recognize protein substrates, and then transfer the ubiquitin from E2 to the targeted protein. This biological process is called ubiquitination, and it is an important biological process which can signal protein degradation via the proteasome. The aim of this protocol is to describe a procedure that determines the level of cellular ubiquitination in a protein-of-interest relative to control cells.
[摘要] 泛素是8.5kDa的蛋白质，其可以分别由泛素激活酶E1和泛素缀合酶E2激活和缀合。 然后泛素E3连接酶识别蛋白质底物，然后将泛素从E2转移到目标蛋白质。 这种生物过程称为泛素化，它是一个重要的生物过程，可以通过蛋白酶体信号蛋白降解。 该方案的目的是描述确定相对于对照细胞的目的蛋白质中的细胞泛素化水平的程序。
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| Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells
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Author:
Date:
2014-04-05
[Abstract] Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the autophagosomal content might be. The method is based on Cyto-ID staining of autophagic compartments (pre-autophagosomes, autophagosomes, and autophagolysosomes) in live cells using Cyto-ID® Autophagy Detection Kit. Autophagic compartments are intermediate constituents of a dynamic lysosomal degradation process and their intracellular abundance at a particular time point is a function of the established equilibrium between their ...
[摘要] 流式细胞术允许非常灵敏和可靠的高通量分析自噬通量。该方法允许在流动中筛选细胞并捕获多组分图像。使用这种技术，可以在悬浮液以及粘附细胞中在胰蛋白酶消化时精确分析自噬流，而与自噬体内容物的异质性无关。该方法基于使用Cyto-ID自动吞噬检测试剂盒的活细胞中的自体吞噬细胞（前自噬体，自噬体和自噬溶酶体）的Cyto-ID染色。自噬区室是动态溶酶体降解过程的中间组分，并且它们在特定时间点的细胞内丰度是其产生和降解之间建立的平衡的函数。自噬吞噬的确定有助于自噬体形成的早期诱导和自噬体成熟的晚期抑制之间的区分，因为两者都导致自噬体存在的最终增加。 Cyto-ID测定基于使用选择性染色自噬隔室的特定染料，因此允许测定自噬通量，作为染色隔室在碱性或活化条件[雷帕霉素（1-5μmol/L），PP242（1- （NH 4 Cl）（10-20μmol/L）或含有6mmol/L葡萄糖（饥饿培养基）的Hanks平衡盐溶液），使用溶酶体化合物阻断自噬溶酶体降解mmol/L）或氯喹（CQ）（5-10μmol/L）。 ΔMFICyto-ID = MFI Cyto-ID（+ CQ/NH 4 Cl）-MFI Cyto-ID（-CQ/NH 4 Cl）。
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