Magnet-assisted Flow Cytometry of in vivo Tumors to Quantitate Cell-specific Responses to Magnetic Iron Oxide Nanoparticles
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Author:
Date:
2020-11-20
[Abstract] A clear understanding of nanoparticle interactions with living systems at the cellular level is necessary for developing nanoparticle-based therapeutics. Magnetic iron oxide nanoparticles provide unique opportunities to study these interactions because of their responsiveness to magnetic fields. This enables sorting of cells containing nanoparticles from in vivo models. Once sorted, flow cytometry can identify individual cell types, which can be further analyzed for iron content, gene or protein expression changes associated with nanoparticle uptake, and for other biological responses at a molecular level. Here we provide a detailed protocol to sort and identify cells in the tumor microenvironment that have internalized magnetic iron oxide nanoparticles following intravenous ...
[摘要] [摘要] 在开发基于纳米颗粒的治疗方法时,必须对纳米颗粒与生物系统在细胞水平上的相互作用有一个清晰的了解。磁性氧化铁纳米颗粒因其对磁场的响应性而提供了研究这些相互作用的独特机会。这使得能够从体内模型中筛选出包含纳米颗粒的细胞。排序后,流式细胞仪可以识别单个细胞类型,然后可以进一步分析其铁含量,与纳米颗粒摄取相关的基因或蛋白质表达变化以及分子水平的其他生物学反应。在这里,我们提供了详细的协议,用于在静脉内给药后分类和鉴定肿瘤微环境中具有内在磁性氧化铁纳米颗粒的细胞。 [背景]几种基于纳米颗粒的抗癌药物已在临床上使用,因为它们已显示出对诊断,抗癌药物传递和热疗的安全性和有效性(Marchal等,2015 ;Wu等,2015)。然而,在我们对癌症纳米医学的理解上仍然存在重大差距,主要是因为纳米颗粒与免疫细胞之间相互作用的细节仍然未知。其他人和我们已经将纳米粒子免疫细胞的相互作用确定为影响体内纳米粒子命运和在肿瘤中保留的关键变量(Sheen等人,2014;Zanganeh等人,2016; ...
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Analyzing the Quenchable Iron Pool in Murine Macrophages by Flow Cytometry
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Author:
Date:
2020-03-20
[Abstract] Tissue-resident macrophages are pivotal for a tightly-regulated iron metabolism at a cellular and systemic level, since subtle iron alterations increase the susceptibility for microbial infections or drive multiple diseases. However, research on cellular iron homeostasis in macrophages remains challenging due to the limited amount of available methods using radioactive 59Fe isotopes or strong iron chelators, which might be inapplicable in certain experimental settings. This protocol describes the analysis of the quenchable iron pool (QIP) in macrophages by loading these cells with exogenous iron-complexes. Thereby, the cytoplasmic iron pool can be determined, since the iron uptake ability of macrophages inversely correlates with intracellular iron levels. Thus, this assay ...
[摘要] [摘要 ] 驻留在组织中的巨噬细胞对于在细胞和全身水平上严格调节铁代谢至关重要,因为细微的铁改变会增加对微生物感染的敏感性或引发多种疾病。然而,由于使用放射性59 Fe同位素或强铁螯合剂的可用方法数量有限,因此巨噬细胞中细胞铁稳态的研究仍然具有挑战性,这在某些实验环境中可能不适用。该协议 描述了通过向这些细胞加载外源铁配合物来分析巨噬细胞中的可淬灭铁池(QIP)。因此,由于巨噬细胞的铁摄取能力与细胞内铁水平成反比,因此可以确定细胞质的铁库。因此,该测定法能够准确分析细胞质铁通量的微小变化,并且几乎适用于所有实验室环境。另外,该方案还可以用于体外和体内的其他免疫细胞类型。
[背景 ] 由于用于全身铁代谢它们的中心调节功能,在多种组织中的巨噬细胞中迅速地改变在不同的刺激的遭遇它们的细胞内的铁水平的微生物感染和疾病(Nairz 等人,2017) 。值得注意的是,大多数细胞中的铁离子被细胞质中的主要铁存储蛋白铁蛋白结合或定位于线粒体或溶酶体等区室(Ma 等,2015 ; Soares和Hamza,2016 ...
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