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 ...
[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] 用一个GFP标记的质粒转染哺乳动物细胞过表达或者调低目的基因是一种最常用的操纵基因表达的方法。想要在细胞循环中检测目的基因的功能,分析GFP阳性细胞和GFP阴性细胞的细胞循环(碘化丙啶,PI染色)是必要的。通常为PI染色用70 %乙醇简单固定细胞淬火GFP信号,在乙醇固定前用低聚甲醛(PFA)固定能帮助染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] 如果你有你所感兴趣的蛋白的抗体,通过使用荧光显微镜检测蛋白定位和相关蛋白丰度的免疫荧光法是一个好方法。使用免疫荧光法的同时能够结合其它的,非抗体荧光染色方法,如使用DAPI标记DNA。
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