| In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells
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Author:
Date:
2016-12-05
[Abstract] To investigate underlying mechanism of chondrogenic hypertrophy, we need proper in vitro hypertrophic model of mesenchymal stem cells (MSCs). This protocol describes our defined method for induction of in vitro chondrogenic hypertrophy of human umbilical cord blood-derived MSCs (hUCB-MSCs). By adding thyroid hormone (T3; triiodothyronine) and minimum osteogenic-inducing factors to culture medium, we could induce hypertrophy of hUCB-MSCs in vitro. Hypertrophic induction was validated using immunohistochemical analysis, Western blotting and reverse transcriptase polymerase chain reaction.
[摘要] 为了研究软骨形成性肥大的基础机制，我们需要适当的体外间充质干细胞（MSC）的增生模型。该协议描述了我们定义的诱导人脐带血衍生的MSC（hUCB-MSC）的体外软骨形成性肥大的方法。通过将甲状腺激素（T3;三碘甲状腺原氨酸）和最小成骨诱导因子添加到培养基中，我们可以在体外诱导hUCB-MSCs的增生。使用免疫组织化学分析，Western印迹和逆转录酶聚合酶链反应验证肥大性诱导。
关键词：间充质干细胞，体外软骨形成性肥大，成软骨分化，三碘甲腺原氨酸;
[背景] ...
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| Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet
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Author:
Date:
2016-01-05
[Abstract] Measurement of the incorporation of radionuclides such as 3H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately quantitate T cell divisions, and an inability to combine proliferation analyses with simultaneous evaluation of the expression of cellular markers in divided cells. By labeling T cells with reactive dyes such as CFSE, Celltrace Violet, and others that are partitioned equally between daughter cells at each cell division, one can relatively easily track generations of proliferated cells and their expression of various molecules by flow cytometry.
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[摘要] 放射性核素如3 H胸腺嘧啶核苷的掺入的测量是用于测定T细胞增殖的经典免疫学技术。然而，这种方法具有超出使用放射性材料的不便之处的缺点，例如体放射性核素结合测量不能准确地定量T细胞分裂，以及不能结合增殖分析与同时评价分裂的细胞标记物的表达细胞。通过用诸如CFSE，Celltrace Violet等活性染料标记T细胞，在每个细胞分裂的子细胞之间平均分配T细胞，可以通过流式细胞术相对容易地追踪增殖细胞的代和它们的各种分子的表达。 > FoxP3 + 调节性T细胞（Treg）是免疫耐受的关键介质，其功能评价是表征许多免疫模型的重要步骤（Rudensky，2011）。基于其表面表达的CD25（Treg：CD4 + CD25 + sup/+），已经分离了经典的CD4 + Treg和常规或"应答者"T细胞， ，Tresp：CD4 + CD25 - sup/- ）。然而，我们和其他人已经注意到，CD4 + CD25 - 细胞群表达FoxP3转录因子并具有抑制功能。因此，我们利用转基因FoxP3-EGFP小鼠促进基于EGFP（并因此FoxP3）表达的抑制基因和应答者群体的活性纯化。在这里我们提出我们适应的协议，用于测定调节性T细胞抑制Celltrace紫罗兰标记响应T细胞。
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| ATP and Lactate Quantification
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Author:
Date:
2013-12-05
[Abstract] Cells use glucose to generate energy by two different metabolic processes: lactic fermentation and aerobic respiration. In the first common series of reactions, glucose is converted into pyruvate. In anaerobic conditions, pyruvate is transformed into lactate, this process yields to 2 ATP molecules per glucose molecule. In the presence of oxygen, pyruvate is imported into mitochondria where it is used in the Krebs (or TCA) cycle and oxydative phosphorylation. The global process of oxydative phosphorylation yields to 32 ATP per glucose molecule. For reasons not fully understood, in some pathological cases like cancer, cells use anaerobic glycolysis even in the presence of oxygen, in which case the process is called aerobic glycolysis (or Warburg effect). This results in an increased uptake ...
[摘要] 细胞使用葡萄糖通过两种不同的代谢过程产生能量：乳酸发酵和有氧呼吸。 在第一个常见的反应系列中，葡萄糖被转化为丙酮酸。 在厌氧条件下，丙酮酸转化为乳酸盐，该过程每个葡萄糖分子产生2个ATP分子。 在氧的存在下，丙酮酸进入线粒体，其用于Krebs（或TCA）循环和氧化磷酸化。 氧化磷酸化的全局过程产生每个葡萄糖分子32个ATP。 由于未完全理解的原因，在一些病理病例如癌症中，细胞即使在氧存在下也使用无氧糖酵解，在这种情况下，该过程称为有氧糖酵解（或沃伯格效应）。 这导致葡萄糖和乳酸盐产生的摄取增加。 细胞内ATP含量和乳酸盐浓度的测量可以提供有氧糖酵解的读出。
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