A Transient Transfection-based Cell Adhesion Assay with 293T Cells
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Author:
Date:
2021-01-05
[Abstract] The in vitro cell adhesion assay is a quantitative method for measuring selective cell adhesion to specific proteins. Traditionally, cell adhesion assays employ purified protein immobilized on a solid glass or plastic surface. Here, we describe a transient 293T cell transfection-based cell adhesion assay to study selective cell adhesion of a specific cell type to a protein of interest. In this protocol, 293T cells are transfected with a mammalian expression plasmid containing mSiglec1 cDNA or an empty plasmid as a mock control and are then cultured to form a monolayer. Subsequently, these Siglec1-expressing and mock-transfected 293T cell monolayers are used for cell adhesion assays with GFP-expressing B16F10 cells. The number of GFP+ cancer cells adhering to each 293T monolayer is a ...
[摘要] [摘要]的体外细胞粘附分析是一种用于测量到特定蛋白选择性细胞粘附的定量方法。传统上,细胞粘附测定采用固定在固体玻璃或塑料表面上的纯化蛋白质。在这里,我们描述了基于瞬时293T细胞转染的细胞粘附试验,以研究特定细胞类型对目标蛋白质的选择性细胞粘附。在该协议中,将293T细胞用包含mSiglec1 cDNA的哺乳动物表达质粒或空质粒作为模拟对照转染,然后培养以形成单层。随后,将这些表达Siglec1和模拟转染的293T细胞单层用于表达GFP的B16F10细胞的细胞粘附测定。GFP +的数量 粘附在每个293T单层上的癌细胞是一种定量手段,用于比较癌细胞与Siglec1的选择性粘附性。该方法消除了表达和纯化目的蛋白以进行体外细胞粘附测定的需要,并且可以容易地用难以纯化的蛋白进行操作,同时保持其天然的原位结构。
关键词:细胞粘附试验,细胞粘附,癌细胞粘附试验,293T,瞬时转染,Siglec1,F荧光显微镜
[背景]细胞-细胞相互作用对于生物学过程,例如组织发育,再生,和临界形态发生,以及免疫应答和癌症转移(Gumbiner,1996 ...
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Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells
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Author:
Date:
2020-12-05
[Abstract] Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, ...
[摘要] [摘要]天然杀伤(NK)细胞是先天性免疫细胞,其特征在于其细胞毒性能力以及活化后的趋化因子和细胞因子分泌。人NK细胞通过CD56表达鉴定。循环的NK细胞可进一步细分为CD56亮(约10%)和CD56暗NK细胞亚群(约90%)。NK细胞样细胞也可以源自人诱导的多能干细胞(iPSC)。为了研究不同的异源NK细胞亚群的趋化因子和细胞因子分泌概况,可以进行细胞内流式细胞仪染色。然而,当起始原料有限时,该测定法具有挑战性。或者,可以通过Luminex测量上清液中分泌的效应子分子来富集,分选,刺激和功能性分析NK细胞亚群。在这里,我们提供了一种快速直接的方案,用于分离和刺激原代NK细胞或iPSC衍生的NK细胞样细胞,并随后检测分泌的细胞因子和趋化因子,这也适用于少量细胞。
[背景]自然杀伤(NK)细胞是先天免疫系统的一部分,提供第一线防御病毒感染和畸形。在人血中,可以基于CD56和CD16表达鉴定出两个不同的NK细胞群体:CD56明亮的CD16 +/-和CD56暗的CD16 + ...
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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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