| Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
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Author:
Date:
2018-07-05
[Abstract] Rat tail collagen solutions have been used as polymerizable in vitro three-dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. These 3D hydrogels are a relatively inexpensive, simple to use model system that can mimic the in vivo physical characteristics of numerous tissues within the body, namely the skin. While confocal imaging techniques such as fluorescence reflection and two-photon microscopy are able to visualize collagen fibrils during 3D imaging without fluorescence, other imaging modalities require direct conjugation of fluorescent dyes to collagen. Here we detail how to generate 3D collagen gels labeled with a fluorescent dye. Furthermore, we go through the steps required to ...
[摘要] 大鼠尾胶原溶液已被用作可聚合的体外三维（3D）细胞外基质（ECM）凝胶，用于单一和集体细胞迁移测定以及球状体形成。 这些3D水凝胶是相对便宜，易于使用的模型系统，其可以模拟体内许多组织（即皮肤）的体内物理特征。 虽然诸如荧光反射和双光子显微镜的共焦成像技术能够在没有荧光的3D成像期间可视化胶原原纤维，但是其他成像模式需要荧光染料直接缀合到胶原。 在这里，我们详细介绍了如何生成用荧光染料标记的3D胶原凝胶。 此外，我们还经历了可重复生成适用于活细胞3D成像技术的明亮胶原水凝胶所需的步骤。
【背景】自20世纪50年代以来，Paul Weiss和Beatrice Garber最初观察到增加血浆浓度（纤维蛋白）对间充质细胞形态的影响（Weiss和Garber，1952），开始研究细胞迁移和细胞与周围微环境的相互作用。在随后的几十年中，生物化学家开始深入研究从鼠尾胶原中纯化提取物，并开始将其用作高度可聚合的3D基质（Fitch et al。，1955; Gross et al。，1955; Chandrakasan et al。，1976）。直到20世纪90年代，3D矩阵才真正对细胞生物学界有用，尤其是研究细胞迁移（Friedl et ...
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| A Technique for the Measurement of in vitro Phospholipid Synthesis via Radioactive Labeling
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Author:
Date:
2016-01-20
[Abstract] This is an assay designed to examine the radioactive phosphorous incorporation when the molecule is being synthesized, which means that only de novo synthesized phospholipids can be detected. Thus, with this technique it is possible to detect in vitro phospholipid synthesis under different required experimental conditions respect to controls (Guido and Caputto, 1990; Ferrero et al., 2014). There are different types of lipids. Among them we can find phospholipids, which contain glycerol esterified with two fatty acyl chains and a phosphate group that can also be bound to an organic molecule that acts as “hydrophilic head”, as shown in Figure 1 for the case of phosphatidylcholine. This structure confers amphipathic properties to lipid molecules that allow them to ...
[摘要] 这是设计用于当合成分子时检查放射性磷掺入的测定，这意味着可以检测到只有新合成的磷脂。因此，利用该技术，可以在相对于对照的不同所需实验条件下检测体外磷脂合成（Guido和Caputto，1990; Ferrero等人，2014） 。有不同类型的脂质。其中我们可以找到磷脂，其含有用两个脂肪酰基链酯化的甘油和磷酸基团，磷酸基团也可以结合到充当"亲水头部"的有机分子，如图1所示的磷脂酰胆碱的情况。这种结构赋予脂质分子两亲性质，使它们形成脂质双层，使磷脂成为生物膜的主要成分。
图1。磷脂结构的代表。摘录自： http：//bio1151.nicerweb .com/Locked/media/ch05/phospholipid.html
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| Measurement of Uptake and Root-to-Shoot Distribution of Sulfate in Arabidopsis Seedlings
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Author:
Date:
2016-01-05
[Abstract] Sulfur is an essential macronutrient required for growth and development of plants. Plants take up sulfate from the soil environment through the function of plasma membrane-bound sulfate transporters expressed at the root surface cell layers. Plants then utilize the incorporated sulfate as the main sulfur source to synthesize sulfur-containing compounds such as cysteine and methionine. Measurement of root sulfate uptake capacity is essential for analyzing mutants showing altered levels of sulfate transporters and/or sulfur metabolic enzymes as a result of genetic modification or due to the effect of intrinsic or environmental factors modulating their gene expression. The method described in this protocol allows quantitative investigation of sulfate uptake rates and root-to-shoot sulfate ...
[摘要] 硫是植物生长和发育所需的必需的大量营养素。植物通过在根表面细胞层处表达的质膜结合硫酸盐转运蛋白的功能从土壤环境中吸收硫酸盐。然后植物利用掺入的硫酸盐作为主要硫源合成含硫化合物，例如半胱氨酸和甲硫氨酸。测量根部硫酸盐吸收能力对于分析由于遗传修饰或由于调节它们的基因表达的内在或环境因素的影响而显示改变的硫酸盐转运蛋白和/或硫代谢酶水平的突变体是必要的。该方案中描述的方法允许使用硫酸盐作为放射性示踪剂定量研究拟南芥幼苗中的硫酸盐吸收速率和根际硫酸盐分布。该方法设计用于在幼苗阶段对多种拟南芥种子，突变体或转基因品系的平行比较。
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