| 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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| Cell Cycle Analysis Using Propidium Iodide Staining with GFP Detection
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Author:
Date:
2012-04-05
[Abstract] Infecting mammalian cells with a GFP construct to overexpress or knockdown target genes is one of the most commonly used methods to study and manipulate gene expression. To determine the target gene function on the cell cycle, analyzing the cell cycle (propidium iodide, PI staining) of GFP positive cells vs GFP negative cells is needed. Usually simple fixation of cells with 70% EtOH for PI staining tends to quench GFP signal; paraformaldehyde (PFA) fixation before ETOH fixation could help to sustain the GFP signal.
[摘要] 用GFP构建体感染哺乳动物细胞以过表达或敲低靶基因是研究和操作基因表达的最常用的方法之一。 为了确定细胞周期上的靶基因功能，需要分析GFP阳性细胞与GFP阴性细胞的细胞周期（碘化丙锭，PI染色）。 通常用70％EtOH的细胞的简单固定用于PI染色倾向于淬灭GFP信号; 多聚甲醛（PFA）固定在ETOH固定之前可能有助于维持GFP信号。
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| Immunofluorescence (Especially for Cells Growing on a Coverglass)
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Author:
Date:
2012-02-20
[Abstract] If an antibody for your protein of interest is available, immunofluorscence is a useful method to detect the localization and relative abundance of the protein by using a fluorescence microscope. Immunofluoresence can be used in combination with other, non-antibody methods of fluorescence staining, for example, the use of DAPI to label DNA. This protocol describes setting up an immunofluorescence experiment using cells grown on a coverglass.
[摘要] 如果您感兴趣的蛋白质的抗体是可用的，免疫荧光是一种有用的方法，通过使用荧光显微镜检测蛋白质的本地化和相对丰度。 免疫荧光可以与其他非抗体荧光染色方法组合使用，例如，使用DAPI标记DNA。 该协议描述了使用生长在盖玻片上的细胞建立免疫荧光实验。
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