| Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells
|
|
Author:
Date:
2017-09-05
[Abstract] Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging. We show that STED increases resolution compared to confocal microscopy, which can be further improved by image processing using deconvolution software.
[摘要] 人肺上皮A549细胞中的呼吸道合胞病毒（RSV）感染诱导丝状伪足，由F-肌动蛋白组成的细胞突起，延伸至相邻的未感染细胞（Mehedi等，2016）。 通过受激发射耗尽（STED）显微镜的高分辨率成像显示沿着这些丝状伪足的丝状RSV颗粒，表明丝状伪足有助于RSV细胞对细胞的扩散（Mehedi等，2016）。 在本协议中，我们描述如何修复，渗透，免疫染色和挂载RSV感染的A549细胞进行STED成像。 我们显示与共聚焦显微镜相比，STED增加了分辨率，可以通过使用去卷积软件的图像处理进一步改进。
【背景】RSV形成多形性病毒颗粒，其长度大约为直径约100nm，长度大约为10μm（Bachi和Howe，1973; Mehedi等，2016）。高分辨率光学显微技术是可视化RSV感染细胞和病毒颗粒之间相互作用的关键。在最近的一项研究中，我们使用超分辨率荧光显微镜来研究人肺上皮A549细胞中的RSV细胞对细胞的扩散。
STED显微镜是超分辨率显微镜技术之一，已被开发以规避约200nm衍射屏障的光限制（Hell和Wichmann，1994; Westphal等人，2008）。 ...
|
|
|
| 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 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。
|
|
|
| Ex vivo Human Natural Killer (NK) Cell Stimulation and Intracellular IFNγ and CD107a Cytokine Staining
|
|
Author:
Date:
2015-06-20
[Abstract] Natural killer (NK) cells comprise 5–20% of peripheral blood mononuclear cells (PBMC) in humans. In addition to their fundamental roles in the defense against viral infections and tumor surveillance, NK cells help shape adaptive immune responses through their production of cytokines. NK cells are traditionally identified as CD3neg, CD14neg, CD19neg lymphocytes expressing CD56. Using a combination of markers that includes CD56 and CD7 greatly increases the ability to define the phenotype and function of NK cell subsets. Two key markers of NK cell function are the production of IFNγ and the release of cytotoxic granules measured by the expression of CD107a. Here we describe a method to assess IFNγ and CD107a expression in NK cells following stimulation with ...
[摘要] 自然杀伤（NK）细胞在人中包含5-20％的外周血单核细胞（PBMC）。 除了它们在防御病毒感染和肿瘤监测中的基本作用，NK细胞通过其细胞因子的产生帮助形成适应性免疫应答。 NK细胞传统上被鉴定为表达CD56的CD3阴性，CD14阳性，CD19阴性淋巴细胞。 使用包括CD56和CD7的标记物的组合极大地增加了定义NK细胞亚群的表型和功能的能力。 NK细胞功能的两个关键标记是IFNγ的产生和通过CD107a的表达测量的细胞毒性颗粒的释放。 在这里我们描述了一种方法来评估在靶细胞或细胞因子刺激后NK细胞中的IFNγ和CD107a表达。 该方法可用于评估来自广泛的研究参与者的外周血单核细胞中NK细胞的一般功能能力。
|
|
|