| 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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| Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells
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Author:
Date:
2017-05-20
[Abstract] Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a ...
[摘要] 抗原特异性T细胞来源的诱导多能干细胞（iPSCs）已显示重新分化为功能性T细胞，从而提供可用于癌症免疫治疗的T细胞的潜在来源。不变性自然杀伤T（Vα24 + iNKT）细胞的人Vα24 + 细胞是T细胞的子集，其特征在于与Vβ11配对的不变Vα24-Jα18的表达，其识别糖脂，如α-半乳糖神经酰胺（α-GalCer），由MHC I类分子CD1d呈递。据报道能够产生IFN-γ的Vα24 + i / KT细胞增加抗肿瘤反应，其影响NK细胞和CD8 +细胞毒性T淋巴细胞以消除MHC - 和MHC + 肿瘤细胞。在这里，我们描述了将人Vα24 + iNKT细胞重编程到iPSC中的鲁棒方案，然后将其重新分化为Vα24 + iNKT细胞（iPS-Vα24功能的iNKT）。我们进一步提供了测定iPS-Vα24 + iNKT细胞活性的方案。背景 以前有报道说，针对晚期非小细胞肺癌（NSCLC）和头颈部癌症的Vα24 + iNKT细胞癌免疫治疗的临床试验显示疗效，耐受性良好（Motohashi et al。等人，2009; Yamasaki等人，2011）。然而，已知来自外周血单核细胞（PBMC）的Vα24 ...
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| ATP and Lactate Quantification
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Author:
Date:
2013-12-05
[Abstract] Cells use glucose to generate energy by two different metabolic processes: lactic fermentation and aerobic respiration. In the first common series of reactions, glucose is converted into pyruvate. In anaerobic conditions, pyruvate is transformed into lactate, this process yields to 2 ATP molecules per glucose molecule. In the presence of oxygen, pyruvate is imported into mitochondria where it is used in the Krebs (or TCA) cycle and oxydative phosphorylation. The global process of oxydative phosphorylation yields to 32 ATP per glucose molecule. For reasons not fully understood, in some pathological cases like cancer, cells use anaerobic glycolysis even in the presence of oxygen, in which case the process is called aerobic glycolysis (or Warburg effect). This results in an increased uptake ...
[摘要] 细胞使用葡萄糖通过两种不同的代谢过程产生能量：乳酸发酵和有氧呼吸。 在第一个常见的反应系列中，葡萄糖被转化为丙酮酸。 在厌氧条件下，丙酮酸转化为乳酸盐，该过程每个葡萄糖分子产生2个ATP分子。 在氧的存在下，丙酮酸进入线粒体，其用于Krebs（或TCA）循环和氧化磷酸化。 氧化磷酸化的全局过程产生每个葡萄糖分子32个ATP。 由于未完全理解的原因，在一些病理病例如癌症中，细胞即使在氧存在下也使用无氧糖酵解，在这种情况下，该过程称为有氧糖酵解（或沃伯格效应）。 这导致葡萄糖和乳酸盐产生的摄取增加。 细胞内ATP含量和乳酸盐浓度的测量可以提供有氧糖酵解的读出。
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