| Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons
|
|
Author:
Date:
2017-09-20
[Abstract] This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over ...
[摘要] 该方案旨在标准化海马和脑皮质神经元原代培养物的体外实验。 培养物应从胚胎发育期间分离的细胞制备，当神经元前体细胞尚未完全分化时。 这有助于提高细胞的质量和数量，同时提供通常在分化的神经元解离期间发生的最小的细胞死亡。 在合适的条件下，在培养皿或多孔板中铺板的细胞将随着时间的推移而发展和建立突触接触，因为神经元培养基提供分化所需的营养和营养因子。 在这个协议中，我们描述了制备皮质和海马神经元培养物的方法。
【背景】本方案描述了使用补充有NeuroCultTM SM1的Neurobasal培养基（Chen等人，2008）的大鼠海马和脑皮层神经元的原代培养物的制备。 NeuroCultTM SM1的组成基于B27补充剂的制剂（Brewer等，1993），但是前者的混合物被发现提高了神经元培养的质量，部分地通过用全转运蛋白替代载脂蛋白转运蛋白 Chen et al。，2008）。 此外，NeuroCultTM SM1的化学成分在原始出版物中有更详细的描述，可以更好地控制实验条件。 用化学确定的培养基制备的神经元培养物的特征在于存在低百分比的星形胶质细胞。 通过添加有丝分裂5-氟-2'-脱氧尿苷的化学抑制剂可以防止维持更长时间的培养物中星形胶质细胞的增殖以允许神经元分化。
|
|
|
| Isolating Taste Buds and Taste Cells from Vallate Papillae of C57BL/6J Mice for Detecting Transmitter Secretion
|
|
Author:
Date:
2016-06-05
[Abstract] Mouse is a well-accepted model for studying taste bud function. Mice readily detect and respond to taste substances that humans consider to have sweet, bitter, salty, sour and umami taste qualities. A great deal of recent research on taste receptors is based on this species. Live mice are needed for these experiments because no alternative in vitro model incorporates all elements of taste transduction and peripheral signaling. The C57BL/6J strain was selected because these mice respond robustly to many taste stimuli and because of variety of transgenic animals, such as PLCβ2-GFP and ...
[摘要] 鼠标是研究味蕾功能的一个被广泛接受的模式。小鼠容易检测和响应人们认为具有甜味，苦味，咸味，酸味和鲜味的品质的味道。最近对味觉感受器的大量研究是基于这个物种。这些实验需要活的小鼠，因为没有替代的体外模型包含所有的味觉转导和外周信号的元件。选择C57BL / 6J菌株，因为这些小鼠对许多味觉刺激反应强烈，并且由于来自该菌株的多种转基因动物，例如PLCβ2-GFP和GAD67-GFP。对C57BL / 6J小鼠进行的行为，神经反应，细胞电生理学和分子生物学的先前分析将为拟议研究奠定坚实的基础（Finger et al。，2005; Huang and Wu，2015; Huang et al。，2007 ）。因此，新鲜安乐死的动物必须用作味蕾的来源，我们将从中分离出味蕾和味道细胞。
|
|
|
| Phagocytosis Assay of Microglia for Dead Neurons in Primary Rat Brain Cell Cultures
|
|
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 ...
[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。
|
|
|