| Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes
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Author:
Date:
2020-04-05
[Abstract] Glucocerebrosidase (GCase) is an important enzyme for the metabolism of glycolipids. GCase enzyme deficiency is implicated in human disease and the efficient measurement of GCase activity is important for evaluating the efficacy of therapeutics targeting this enzyme. Existing approaches to measure GCase activity include whole blood mass spectrometry-based assays, where an internal standard is used to measure the accumulation of ceramide following metabolism of the synthetic substrate C12-glucocerebroside, and the utilisation of fluorescent probes that bind active GCase and/or release fluorescent metabolites upon cleavage by GCase. Here, we describe the application of a fluorescence-activated cell sorter-based assay to efficiently quantitate GCase enzyme activity in the monocyte population ...
[摘要]
[摘要 ] 葡萄糖脑苷脂酶（GCase ）是糖脂代谢的重要酶。GCase 酶缺乏症与人类疾病有关，GCase 活性的有效测量对于评估靶向该酶的治疗剂的功效至关重要。现有的测量GCase 活性的方法包括基于全血质谱的分析，其中使用内标测量合成底物C12-葡萄糖脑苷代谢后神经酰胺的积累，以及利用结合活性GCase 和// 的荧光探针的利用。或通过GCase 裂解后释放荧光代谢物 。在这里，我们描述了基于荧光激活细胞分选术的测定方法的应用，以有效地定量人类外周血单核细胞单核细胞群中的GCase 酶活性。细胞渗透性的GCase 衬底5-（Pentafluorobenzoylamino ）荧光素二-β-D- 吡喃葡萄糖苷（PFB- FDGlu ）提供了一个用于测量的GCase 活性，由此酶裂解产生的绿色荧光PFB-F染料，在FL-检测流式细胞仪的1个通道。使用溶酶体GCase 活性抑制剂，conduritol B-环氧，以确保特异性。该协议为测量活的单个细胞中的GCase 活性提供了一种有利的方法。
[背景 ] 葡萄糖脑苷脂酶（GCase ），由 GBA1基因是一种溶酶体水解酶，可将葡糖神经酰胺转化为葡萄糖和神经酰胺。GBA1 ...
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| Contemporaneous Measurement of Outer and Inner Membrane Permeability in Gram-negative Bacteria
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Author:
Date:
2020-03-05
[Abstract] The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the ...
[摘要] [摘要 ] 细菌中多药耐药性的出现和迅速扩散导致对新型抗菌剂的迫切需求。膜通透性是许多针对革兰氏阴性细菌的抗菌分子的机制。因此，为了确定潜在的抗菌分子的功效，重要的是评估治疗后细菌膜通透性的变化。这项研究描述了使用荧光探针N-苯基-1-萘胺和碘化丙啶测定外膜和内膜通透性的方案。与其他实验（例如电子显微镜和最小杀菌浓度的测定）相比，该方法提供了一种更简单，更快速且经济高效的方法来评估抗菌分子的膜通透性和杀菌功效。这项研究提出了一种相对于传统方案的优化方案，该方案是在与抗菌测定相同的培养条件下，将细菌与抗菌分子一起孵育，然后在不含肉汤和抗菌分子的缓冲液中检测荧光探针的信号。此协议可避免的效果养分缺乏对细菌的生理状态和抗菌molecul的干扰ES 朝向的F luorescent探针。因此，该方法可以有效，准确地评估膜的渗透性，并与从其他抗菌测定法（如最小抑菌浓度和时间杀灭曲线测定法）获得的结果相匹配。
[背景 ] ...
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| Platelet Migration and Bacterial Trapping Assay under Flow
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Author:
Date:
2018-09-20
[Abstract] Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing efficient intravascular bacterial trapping. Here, we describe a method that allows analyzing platelet migration in vitro, focusing on their ability to collect bacteria and trap bacteria under flow.
[摘要] 血小板对于止血和血栓形成至关重要，但在免疫反应中也起着不同的作用。 我们最近报道了血小板在体外体外和体内感染部位迁移。 重要的是，血小板利用它们迁移的能力来收集和捆绑纤维蛋白（ogen）结合的细菌，从而实现有效的血管内细菌捕获。 在这里，我们描述了一种方法，允许分析血小板在体外的迁移，重点是它们收集细菌和捕获流动细菌的能力。
【背景】血小板是从巨核细胞释放的小的无核细胞片段，其存在于哺乳动物生物的骨髓内（Machlus和Italiano，2013）。大约7500亿血小板在人体血液中循环，不断扫描脉管系统以破坏内皮表面。在遇到内皮损伤时，血小板立即被招募在充分表征的事件级联中，包括初始血小板束缚和滚动，然后是血小板活化，粘附和扩散，最终导致纤维蛋白（ogen）依赖性聚集和随后的血栓收缩（Jackson， 2007）。血小板栓塞形成是生理性止血的主要步骤，但也是动脉粥样硬化斑块破裂后的病理性血栓形成，触发心肌梗塞或中风（Jackson，2011）。
除了在止血和血栓形成中的公认作用外，血小板还发展出多种免疫功能（Semple et al。，2011）。作为第一批招募炎症和感染部位的细胞，血小板在启动血管内免疫反应中起着重要作用（Wong et ...
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