| Acute Live/Dead Assay for the Analysis of Toxic Effects of Drugs on Cultured Neurons
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Author:
Date:
2016-08-05
[Abstract] The primary culture of central nervous system (CNS) neurons is a popular test system for a rapid, quantitative and reliable assessment of the effects of drugs on central neurons. Consequently, studies on the excitotoxicity of NMDA activation and on intracellular calcium handling machineries with respect to ischemic damage to the brain as well as neurodegenerative diseases have been highly productive (Ankarcrona et al., 1995). This created the need to establish a standard method for assessment of neurotoxicity. Several methods are currently being used, including LDH leakage and MTT assays (Mosmann, 1983; Decker and Lohmann-Matthes, 1988). We have used another common method for assessing acute cell death, the dead/live assay (Slepian et al., 1996). It provides a precise ...
[摘要] 中枢神经系统（CNS）神经元的主要培养物是用于快速，定量和可靠地评估药物对中枢神经元的影响的流行测试系统。因此，关于NMDA激活和细胞内钙处理机制对脑缺血性损伤以及神经退行性疾病的兴奋性毒性的研究已经很有成效（Ankarcrona等，1995）。这就需要建立一种评估神经毒性的标准方法。目前正在使用几种方法，包括LDH渗漏和MTT测定（Mosmann，1983; Decker和Lohmann-Matthes，1988）。我们使用另一种常见的方法来评估急性细胞死亡，死亡/活体检测（Slepian等，1996）。它提供了暴露于有毒物质后细胞死亡过程的精确时间和浓度评估，在我们的情况下，以前提出用作选择性PKM-zeta拮抗剂的ζ抑制肽（ZIP）（Ling等， 2002; Pastalkova等人，2006; Sadeh等人，2015）。在该测定中，我们用Calcein-AM负载细胞，其在穿透到活神经元中时，从非荧光化合物转化为高度荧光的绿色荧光团。随后，在渗透死细胞的碘化丙啶（PI）和计数红/绿荧光细胞的存在下，我们将神经元暴露于不同浓度的ZIP各种持续时间。这种方法可以让我们检查哪些细胞是活的，暴露在有毒物质之后死亡，以及细胞死亡的时间过程。
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| 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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