| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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| Phagolysosomal Trafficking Assay
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Author:
Date:
2014-07-05
[Abstract] Phagolysosomal trafficking is an important innate defense pathway that clears microbes by delivering them to lysosomes, the degradative compartment of the cell. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, subverts this host defense mechanism by arresting maturation of the phagosome. The ability of Mtb to arrest its delivery to the lysosome can be demonstrated by the prolonged co-localization of bacteria containing phagosomes/vacuole with early phagosomal markers [such as, Ras- related proteins in the brain 5 (Rab5) and Transferrin receptor (TfR)], and a failure to acquire late phagosomal and lysosomal markers (such as Rab7 and LAMP1) (Deretic and Fratti, 1999, Mehra et al., 2013). Here, a protocol is outlined for infection of macrophages with ...
[摘要] 吞噬溶酶体运输是重要的先天防御途径，通过将其递送至溶酶体，即细胞的降解区室来清除微生物。结核病的致病因子结核分枝杆菌（Mtb）通过阻滞吞噬体的成熟来破坏这种宿主防御机制。 Mtb阻止其递送至溶酶体的能力可以通过含有吞噬体/液泡的细菌与早期吞噬体标记物[例如，脑5中的Ras相关蛋白（Rab5）和转铁蛋白受体（TfR） ）]，以及未获得晚期吞噬体和溶酶体标记物（例如Rab7和LAMP1）（Deretic和Fratti，1999，Mehra等人，2013）。在这里，概述了用分枝杆菌物种感染巨噬细胞的方案，所述分枝杆菌物种如致病性Mtb疫苗菌株牛分枝杆菌 - 卡介苗（BCG）和快速分裂的非致病性耻垢分枝杆菌（Msmeg），然后间接免疫荧光显微镜观察宿主液泡标记。此后，通过使用数学工具处理细菌的二值图像来进行分枝杆菌和宿主液泡标记如TfR和LAMP1之间的共定位程度的自动定量。这导致直接在细菌/细菌簇周围的这些宿主标志物的平均荧光强度（MFI）的定量，相对于手动完成时具有增加的灵敏度。通过操纵宿主或病原体，该测定可用于评价细胞内运输的宿主或细菌决定簇。基本方法可以应用于研究其他细菌或颗粒状珠子的运输，尽管感染和吞噬体成熟的动力学将取决于吞噬性货物。数学分析工具可用于许多标准成像分析程序。然而，对于类似分析的任何适应性应由个体用户利用其成像和分析平台来确认。
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| Lysosomal Stability Assay
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Author:
Date:
2014-06-20
[Abstract] This assay makes use of the dye Acridine Orange (AO) to determine the stability of lysosomes in living cells upon exposure to a confocal microscope laser.
AO is a lipophilic amine that readily diffuses into cells (Figure 1). Inside the cell it enters the acidic lysosomal compartment where it is protonated and sequestered, shifting its emission spectrum towards a longer wavelength (i.e. red). Once inside the lysosomes, the metachromatic AO sensitizes the lysosomal membrane to photo-oxidation by blue light (Brunk et al., 1997). Upon light-induced loss of the lysosomal pH gradient and subsequent leakage of AO into the cytosol, the emission spectrum of AO shifts from red to green (Figure 2). Hence, loss of lysosomal integrity can be measured as a ‘loss of red ...
[摘要] 该测定利用染料吖啶橙（AO）确定在暴露于共焦显微镜激光器时活细胞中溶酶体的稳定性。
图1。吖啶橙
AO是一种易于扩散入细胞的亲脂胺（图1）。在细胞内部，它进入酸性溶酶体区室，在那里它被质子化和螯合，将其发射光谱移向更长的波长（即，红色）。一旦在溶酶体内，异染色质AO使溶酶体膜通过蓝光的光氧化敏感（Brunk等人，1997）。在光诱导的溶酶体pH梯度的损失和随后的AO向胞质溶胶中的泄漏后，AO的发射光谱从红色变为绿色（图2）。因此，溶酶体完整性的损失可以被测量为"红点的丢失"或作为绿色荧光的定量升高（Petersen等人，2010; Kirkegaard等人，，2010; Petersen ，2013）。
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