| Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays
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Author:
Date:
2021-03-05
[Abstract] An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary ...
[摘要] [摘要]内源性生物钟系统使生物能够适应与时间相关的环境变化。结果，大多数生理过程表现出例如能量代谢，免疫功能，睡眠或激素产生的每日节律。下丘脑生物钟已被确认在协调许多这些过程中起特定作用。 原代神经元文化被广泛用作研究神经元内分子事件的生理相关模型。然而，由于昼夜节律包括较长时间范围内的动态分子变化，而这种变化在各个细胞之间会有所不同，因此纵向测量方法对于研究下丘脑神经元昼夜节律的调节至关重要。在这里，我们提供了用于生成表达昼夜节律性荧光素酶报道基因的下丘脑神经元文化的协议。通过执行生物发光测量，此类报告细胞可用于以高时间分辨率纵向监测细胞昼夜节律。
[背景]为了适应重复在其环境中的时间-日期依赖性变化，许多生物已开发出一种内源性生物钟系统调节行为和生理过程的24小时的节律（夏尔马，2003）。在哺乳动物中，一个昼夜节律性起搏器主要位于下丘脑上视交叉上核（SCN）。它与外部时间协调整个身体的细胞时钟调节。睡眠，食欲和新陈代谢的每日模式由下丘脑神经元中的细胞昼夜节律调节（Cedernaes等，2019）。
在哺乳动物细胞中，昼夜节律时钟由互锁的转录-翻译反馈环（TTFL）组成。在核心TTFL中，转录因子昼夜运动输出周期kaput（CLOCK）和脑和肌肉芳基碳氢化合物受体核转运蛋白样蛋白1（BMAL1或ARNTL）激活其自身阻遏物，周期（PER1-3）和隐色蛋白的表达（CRY1 ...
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| Generation of Neuron-enriched Cultures (Method 2)
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Author:
Date:
2011-11-05
[Abstract] Neuron-enriched cultures are a useful tool to study neuronal development and the molecular pathways that come in to play during neuronal death in various neurological disorders. This protocol is for generating midbrain neuronal cultures from late embryo rodent brain. This method uses Neurobasal medium and B27 serum-free supplement. The long-lasting (> 4 weeks) midbrain neuron-enriched cultures generated following this protocol have been wildly used to study the pathogenesis of Parkinson’s disease, the most common neurodegenerative movement disorder. The neuron-enriched cultures prepared following this protocol will contain < 10% astroglia. the usage of b27 serum-free supplement will definitively increase the cost. this protocol has been developed and improved over the years by various researchers in dr. hong’s lab, especially dr. bin liu. 10%="" astroglia.="" the="" usage="" of="" b27="" serum-free="" supplement="" will="" definitively="" increase="" the="" cost.="" this="" protocol="" has="" been="" developed="" and="" improved="" over="" the="" years="" by="" various="" researchers="" in="" dr.="" hong’s="" lab,="" especially="" dr.="" bin=""> ...
[摘要] 神经元富集的培养物是研究神经元发育和在神经元死亡期间在各种神经疾病中发挥的分子途径的有用工具。 该协议是用于从晚期胚胎啮齿动物脑产生中脑神经元培养物。 该方法使用Neurobasal培养基和B27无血清补充剂。 根据该方案产生的持续（> 4周）中脑神经元富集培养物已被广泛用于研究帕金森病的发病机理，帕金森病是最常见的神经退行性运动障碍。 按照该方案制备的富含神经元的培养物将含有<10％星形胶质细胞。 使用b27无血清补充剂将明显增加成本。="">
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| Generation of Neuron-enriched Cultures (Method 1)
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Author:
Date:
2011-11-05
[Abstract] This protocol will guide you through the process for generating midbrain neuronal cultures from late embryo mouse brain. These cultures serve as a useful tool to study molecular pathways during neuronal death in various neurological disorders. This method uses cytosine β-D-arabinofuranoside in the cultures to suppress the proliferation of glial cells. Seven days after the seeding, the neuron-enriched cultures prepared following this protocol will contain less than 10% glia cultures. The concentrations of β-D-arabinofuranoside should be adjusted according to your culture condition. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.
[摘要] 这个协议将指导您通过从晚胚胎小鼠大脑产生中脑神经元文化的过程。 这些文化作为一个有用的工具，研究神经元死亡期间各种神经系统疾病的分子途径。 该方法在培养物中使用胞嘧啶β-D-阿拉伯呋喃糖苷抑制神经胶质细胞的增殖。 接种后7天，按照该方案制备的富含神经元的培养物将含有少于10％的胶质细胞培养物。 应根据您的培养条件调整β-D-阿拉伯呋喃糖苷的浓度。 这个协议已经由Hong博士实验室的各种研究人员，尤其是刘博士多年来开发和改进。
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