| Phagocytosis Assay of Microglia for Dead Neurons in Primary Rat Brain Cell Cultures
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Author:
Date:
2016-04-20
[Abstract] Clearance of dead brain tissue including the dead neurons through phagocytosis is an endogenous function of microglia in the brain, which is critical for inflammation resolution after ischemic stroke or head trauma. By regulating the function or polarization status of microglia, we may control their phagocytosis efficacy and therefore the cleanup process for the dead brain tissue. We cultured rat cortical neurons and microglia from the same litter of embryos. The cultured neurons are subjected to irradiation for inducing neuronal apoptosis. After labeling with propidium iodide (PI), the dead neurons (DNs) are exposed to the cultured microglia for phagocytosis assay. By counting the number of DNs in each microglia, we calculate the phagocytosis index to quantify the phagocytosis efficacy ...
[摘要] 通过吞噬作用来清除包括死亡神经元在内的死亡脑组织是脑中小胶质细胞的内源性功能，这对缺血性卒中或头部创伤后的炎症分解至关重要。通过调节小胶质细胞的功能或极化状态，我们可以控制其吞噬功效，从而控制死脑组织的清除过程。我们从相同的胚胎培养大鼠皮质神经元和小胶质细胞。培养的神经元经受辐射诱导神经细胞凋亡。用碘化丙啶（PI）标记后，将死亡神经元（DN）暴露于培养的小神经胶质细胞进行吞噬试验。通过计算每个小胶质细胞中的DN数量，我们计算吞噬指数，以量化小胶质细胞对DN的吞噬功效。方案分为4个部分：A）从产前大鼠胚胎培养大鼠皮质神经元，B）将死亡神经元作为吞噬作用靶标，C）培养大鼠脑小胶质细胞，D）定量小神经胶质细胞向死亡神经元的吞噬指数。
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| Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices
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Author:
Date:
2016-02-05
[Abstract] An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (Warburg, 1923), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis. In our approach, we maintain freshly resected tissue slices (both cancerous and non- cancerous from the same organ of the same subject) in cell culture media, and treat with appropriate stable isotope-enriched nutrients, e.g., 13 ...
[摘要] 这种方法的一个重要组成部分是评估肿瘤微环境对肿瘤生长和存活的作用。为了解决这个问题，我们采用Warburg（Warburg，1923）的原始方法，通过将薄组织切片与稳定同位素解析代谢组学（SIRM）组合来确定人体组织的详细代谢活性。 SIRM使得能够在定义的时间段内跟踪诸如葡萄糖或谷氨酰胺的来源分子的代谢转化，并且是详细的途径追踪和通量分析的要求。在我们的方法中，我们保持在细胞培养基中新鲜切除的组织切片（来自同一受试者的相同器官的癌性和非癌性），并用适当的稳定的富含同位素的营养物，例如葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖， - 谷氨酰胺。这些切片可存活至少24小时，并使得有可能消除对靶组织代谢的系统影响，同时保持原始的3D细胞结构。因此，它是用于评估治疗剂对靶组织代谢的影响及其对个体患者的治疗功效的极好的临床前平台（Xie等人，2014; Sellers等人，/em>，2015）。
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