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DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)

DAPI(4'',6-二脒基-2-苯基吲哚,二盐酸盐)

Company: Thermo Fisher Scientific
Catalog#: D1306
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Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines
Author:
Date:
2018-03-20
[Abstract]  The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells. [摘要]  主要纤毛是一种非运动感觉细胞器,其装配和拆卸与细胞周期进程密切相关。 初级纤毛在静止细胞中从基体拉长并随着细胞重新进入细胞周期而被吸收。 睫状动力失调与纤毛病和其他人类疾病有关。 体外血清刺激的睫状体装配/分解测定已经在解决睫状动力学中感兴趣的蛋白质的功能方面受到欢迎。 在这里,我们描述了转染人视网膜色素上皮细胞(RPE-1)和对转染细胞进行睫状体装配/分解测定的充分测试的方案。

【背景】初级纤毛是毛发样感觉细胞器,其在G 0 / G 1期出现,并且在细胞周期的S期之前分解(Tucker等, et al。,1979)。先前的研究已经证实,某些未转化的细胞类型(即,甚至是RPE-1细胞,3T3成纤维细胞和小鼠胚胎成纤维细胞[MEFs])可以被饿死以诱导静止和睫状体形成。随后的血清再次添加触发双相睫状体吸收,其在刺激后2小时和24小时达到峰值(Tucker等人,1979; Li等人,2011) 。该现象为文献中常用的血清刺激的睫状体组装/分解测定奠定了基础,以鉴定参与睫状体组装和拆卸的蛋白质(Pugacheva等人,2007; ...

Mouse Satellite Cell Isolation and Transplantation
Author:
Date:
2018-01-20
[Abstract]  Satellite cell (SC) transplantation represents a powerful strategy to investigate SC biology during muscle regeneration. We described here a protocol for SC isolation from green fluorescent protein (GFP)-expressing mice and their transplantation into murine muscles. This procedure was originally used to assess the effects of the hormone unacylated ghrelin on muscle regeneration, in particular evaluating how the increase of unacylated ghrelin in the recipient muscle affected the engraftment of donor SCs (Reano et al., 2017). [摘要]  卫星细胞(SC)移植代表了肌肉再生期间SC生物学研究的强大策略。 我们在这里描述了从绿色荧光蛋白(GFP)表达的小鼠和他们的小鼠肌肉移植SC分离的协议。 该程序最初用于评估激素非酰化生长素释放肽对肌肉再生的影响,特别是评估受体肌肉中未酰化的生长素释放肽的增加如何影响供体SC的植入(Reano等人,2017年)。

【背景】由分化的肌纤维组成的骨骼肌可以在受伤时再生。肌肉再生依赖于居住在肌肉纤维基底层之下的称为卫星细胞(SC)的静止驻留干细胞群(Mauro,1961)。受伤后,SC经历激活,广泛的增殖,分化和融合,最终修复或替换受损的肌纤维(Collins等人,2005)。

由于移植的成肌细胞可以与宿主成肌细胞融合,因此SC的移植被认为是杜氏肌营养不良症(DMD)的潜在疗法多年的时间,这提示了在缺陷纤维中功能性修复的可能性(Partridge ...

FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand
Author:
Date:
2017-12-20
[Abstract]  Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and ... [摘要]  从新鲜组织中直接分离人类神经和胶质瘤干细胞允许其在没有事先培养的情况下进行生物学研究,并且可以在其天然状态中捕获其分子表型的新方面。最近,我们展示了前瞻性地从新鲜人类生发基质和胶质母细胞瘤样品中分离干细胞群的能力,利用细胞在荧光激活细胞分选(FACS)中结合表皮生长因子(EGF)配体的能力。我们证明FACS分离的EGF结合的神经和成胶质细胞瘤细胞群体在体外包含球体形成的集落,并且能够自我更新和多向分化。在此我们详细描述了具有来自新鲜死亡和手术组织的干细胞特性的EGF-结合(即EGFR +)人类神经和胶质瘤细胞的纯化方法。利用天然配体结合能力前瞻性分离干细胞群的能力为了解非培养条件下的正常和肿瘤细胞生物学打开了新的门,并且适用于在种群和单细胞分辨率下的各种下游分子测序研究。

【背景】由于缺乏通用的神经和神经胶质瘤干细胞标志物(Lathia et al。,2015)以及频繁依赖于培养的细胞,理解人神经和胶质瘤干细胞的内在生物学一直是一个挑战比那些直接从组织分离的。跨膜糖蛋白Prominin或CD133是分离神经(Uchida等,2000)和神经胶质瘤干细胞(GSC)(Singh等,2000)的最好描述和经常使用的干细胞标记物之一。等人,2003; Singh等人,2004; ...

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