Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells
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Author:
Date:
2017-09-20
[Abstract] Anthracyclines, such as doxorubicin and daunorubicin, are DNA damaging agents that autofluoresce and can be readily detected in cells. Herein, we developed suitable assays to quantify and localize daunorubicin in mammalian cells. These assays can be exploited to identify components that are involved in the uptake of anthracyclines.
[摘要] 蒽环类药物,如多柔比星和柔红霉素,是DNA损伤剂,其可自发荧光并且可以容易地在细胞中检测到。 在这里,我们开发了合适的测定法来定量和定位哺乳动物细胞中的柔红霉素。 可以利用这些测定来鉴定参与蒽环类药物吸收的成分。 【背景】蒽环类药物,如多柔比星和柔红霉素,通过损伤DNA起作用,用于治疗各种类型的癌症,包括急性骨髓性白血病。当癌症患者被系统地给予蒽环类抗生素时,有几个因素限制了到达肿瘤部位的药物的量(Chauncey,2001; Deng等,2014; Riganti等,2015)。在肿瘤中,对癌细胞的药物活性受到药物吸收不良,药物流出过量以及细胞靶标变化的限制。几十年来,这些DNA损伤剂如何进入癌细胞还不清楚(Aouida等,2010; Cesar-Razquin等,2015; Zhang等,2015)。为了解决这个问题,我们开发了三种可靠的体外测定法来监测柔红霉素在细胞中的积累。这些测定中的两个是定量的,需要获得荧光活化细胞分选(FACS)口径和Fluoroskan仪器,第三个是使用落射荧光显微镜进行半定量。使用这些测定法,我们确定柔红霉素以时间和浓度依赖的方式进入细胞,并且每种细胞类型显示不同的摄取速率,这表明活性过程参与蒽环类药物的摄入(Andreev等人, ...
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DNA Damage Induction by Laser Microirradiation
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Author:
Date:
2016-12-05
[Abstract] Genome instability can lead to cell death, senescence and cancerous transformation. Specific repair pathways have evolved to prevent accumulation of DNA lesions. Studying these highly dynamic and specific repair pathways requires precise spatial and temporal resolution, which can be achieved through a combination of laser microirradiaiton and live cell microscopy. DNA lesions are introduced at pre-determined sub-nuclear sites and repair can be analyzed in real time in living cells when using fluorescently tagged repair proteins (Mortusewicz et al., 2008). Alternatively, laser microirradiation can be combined with immunofluorescence analysis to study recruitment of endogenous proteins to laser-induced DNA damage tracks that can be visualized by positive controls like, e.g., ...
[摘要] 基因组不稳定性可导致细胞死亡,衰老和癌性转化。特异性修复途径已经进化以防止DNA损伤的累积。研究这些高度动态和特定的修复途径需要精确的空间和时间分辨率,这可以通过激光微激光和活细胞显微镜的组合实现。当使用荧光标记的修复蛋白时,在预定的亚核位点引入DNA损伤并且可以在活细胞中实时分析修复(Mortusewicz等人,2008)。或者,激光微辐照可与免疫荧光分析结合以研究内源蛋白质对激光诱导的DNA损伤轨迹的募集,其可通过阳性对照例如标记DNA断裂位点的γH2AX显现。 关键字:微辐射,活细胞成像,DNA损伤,DNA修复,DNA损伤,DNA损伤反应,免疫荧光,显微镜等 /strong>哺乳动物细胞的基因组完整性不断受到通过外部和内部来源引入的DNA损伤的挑战。最常见的DNA损伤是氧化碱基,双链断裂,单链断裂,链间和链内交联和UV加合物。已经发展了各种DNA损伤信号传导和修复途径以处理这些损伤。为了使DNA修复快速,精确和有效,涉及感测,信号传导和修复特定DNA损伤的许多蛋白质必须在空间和时间上协调。此外,DNA被组织成更高级的染色质结构,因此对于DNA损伤,DNA修复酶是可及的,染色质必须重塑。激光微照射与高级活细胞显微镜相结合允许在活细胞的上下文中研究这些高度动态的过程(Mortusewicz等人,2008)。这里描述的协议使用405 ...
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Immunolocalization of Proteins in Corals: the V-type H+-ATPase Proton Pump
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Author:
Date:
2015-09-05
[Abstract] Here we describe the immunolocalization of a membrane-bound proton pump, the V-type H+-ATPase (VHA), in tissues and isolated cells of scleractinian corals. Immunolocalization of coral proteins requires additional steps not required for various model organisms, such as decalcification of the coral skeleton for immunohistochemistry or removal of cells away from the skeleton for immunocytochemistry. The tissue and cell preparation techniques described here can be adapted for localization of other coral proteins, provided the appropriate validation steps have been taken for the primary antibodies and species of coral used. These techniques are important for improving our understanding of coral cell physiology
[摘要] 在这里我们描述膜结合质子泵,V型H + -ATPase(VHA),在组织和分离的细胞的scleractinian珊瑚的免疫定位。 珊瑚蛋白的免疫定位需要对于各种模型生物不需要的额外步骤,例如用于免疫组织化学的珊瑚骨架的脱钙或从用于免疫细胞化学的骨架移除细胞。 本文所述的组织和细胞制备技术可适用于其它珊瑚蛋白的定位,条件是已对所用的一级抗体和珊瑚种类采取了适当的验证步骤。 这些技术对于提高我们对珊瑚细胞生理学的理解是重要的
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