| Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma
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Author:
Date:
2017-09-05
[Abstract] Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further ...
[摘要] 大多数上皮肿瘤已经显示含有可能是肿瘤进展和转移的驱动力的癌症干细胞（Kreso和Dick，2014; Nassar和Blanpain，2016）。 为了深入研究这些细胞，依赖于细胞表面标志物或荧光报告基因的细胞分离策略是必不可少的，分离策略必须保持其活力。 从大部分肿瘤中分离不同细胞群的能力将继续加深我们对癌症干细胞生物学的认识。 在这里，我们报告了结合机械肿瘤解离，酶处理和流式细胞术的策略，从其天然微环境中分离出纯种群的上皮癌干细胞。 该技术可用于进一步功能性地分析癌症干细胞（RNA测序和表观遗传学分析），在培养物中培养它们或在移植测定中直接使用它们。
【背景】肿瘤复发和转移是大多数与癌症有关的死亡的主要原因。恶性肿瘤可能由干细胞群体启动和维持（Nassar和Blanpain，2016; Bonnet和Dick，1997），这些细胞是预防复发的重要治疗靶点（Baumann et al。，2008）。研究表明，鳞状细胞癌由肿瘤干细胞亚群维持，其抗药性，并通过进行自我更新和分化（如正常干细胞）引发肿瘤复发，产生增殖祖细胞，其分化形成肿瘤大部分（Locke et al。，2005; Prince et al。，2007; Malanchi et al。，2008; de Sousa e Melo et ...
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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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| Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish
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Author:
Date:
2016-10-05
[Abstract] The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used MitoSOX and CellROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses.
[摘要] 自由基的产生是正常细胞代谢的结果。 自由基涉及无数不同的细胞和生物学功能，例如信号传导，增殖，细胞死亡，衰老，炎症等。 在生理条件下，活性氧（ROS）的水平被细胞严格调节。 然而，在应激条件例如氧化应激期间，ROS水平增加导致对不同分子如DNA，脂质和蛋白质的损伤。 增加的ROS水平与不断增加的不同疾病列表相关联。 在这个协议中，我们使用MitoSOX和CellROX绿色氧化应激探针标记细胞内ROS和使用细胞分选和共聚焦分析检测荧光。
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