| Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays
|
|
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 ...
[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 ...
|
|
|
| Generation of Neuron-enriched Cultures (Method 2)
|
|
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 ...
[摘要] 神经元富集培养是来研究在神经元死亡中各种神经系统疾病的分子通路的有用工具。本方法可用于从啮齿动物后期胚胎大脑获得中脑神经元培养。本方法使用神经基质培养基以及B27无血清补充物。 本实验方法所获得的长期（大于四周）中脑神经元富集培养已广泛用于研究帕金森氏病这一最常见的神经退化性运动障碍疾病的发病机制。本方法准备的神经元富集将含有<10％的星形胶质细胞。使用b27无血清补充物将肯定会增加成本。此实验方法经dr. hong实验室不同研究人员多年的发展和完善，尤其是dr. bin liu. hong实验室不同研究人员多年的发展和完善，尤其是dr.="" bin="">
|
|
|
| Generation of Neuron-enriched Cultures (Method 1)
|
|
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 ...
[摘要] 本方法将引导您完成从后期胚胎小鼠大脑产生中脑神经元培养的整个过程。这种培养是来研究在神经元死亡中各种神经系统疾病的分子通路的有用工具。这种方法使用胞嘧啶β-D-arabinofuranoside（阿糖胞嘧啶）到培养基中，抑制胶质细胞增生。播种后第7天，在本方法后的神经元富集的准备将含有不少于10％的神经胶质细胞培养基。β-D-arabinofuranoside的浓度应根据培养基条件调整。此实验方法经Dr. Hong实验室不同研究人员多年的发展和完善，尤其是Dr. Bin Liu.
|
|
|