In vivo Mouse Mammary Gland Formation
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Author:
Date:
2020-07-05
[Abstract] For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several In vitro assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for in vivo mammary gland formation assay, including preparation of “cleared” recipients and mammary cells for implantation, the surgery process and ...
[摘要] [摘要 ] 多年来,乳腺一直是研究成体干细胞的自我更新和分化以及这些过程的调控机制的完美例证。为了评估给定的基因和/或其他因素对乳腺细胞的干性,有几个函数体外测定法开发出来,如微球体由mRNA表达形成试验,干细胞标志物的检测或流式细胞术等。然而,在雌性小鼠清除的脂肪垫中整个坐骨的重建能力是估计细胞干性的黄金标准。在这里,我们描述了体内的分步操作方案 乳腺形成测定,包括准备“清除”的受体和用于植入的乳腺细胞,手术过程以及如何评估实验结果。结合通过基因编辑和/或药物处理对乳腺细胞的操作,该方案在乳腺干细胞和乳腺发育的研究中可能非常有用。
[背景 ] 作为哺乳动物最典型的器官之一,乳腺(MG)是外分泌腺,负责泌乳。MG的发育受某些性激素的控制,这些激素的水平精确地调节了MG在不同发育阶段的结构,细胞组成和功能变化(Henigighausen and Robinson,2005)。许多遗传和环境因素都参与了乳腺干细胞的调控和MG的发育。为了研究这些因素的功能和机理,已经开发了几种方法,特别是用于评估乳腺细胞的干性。先前的研究表明,只有MG的基底细胞而非管腔细胞能够在受体雌性小鼠清除的脂肪垫中重建上皮树,这表明乳腺干细胞仅存在于基底谱系中(Van Keymeulen 等,2011)。 )。后来,包括我们在内的许多研究发现了乳腺干细胞的几种标志物(Prater ...
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Investigating Neural Stem Cell and Glioma Stem Cell Self-renewal Potential Using Extreme Limiting Dilution Analysis (ELDA)
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Author:
Date:
2018-09-05
[Abstract] Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in ...
[摘要] 作为神经球生长的神经胶质干细胞(GSC)表现出与作为神经球生长的神经干细胞(NSC)相似的特征,包括自我更新和分化的能力。 GSC被认为在癌症的发生和发展中起作用。 GSC的自我更新潜力被认为反映了与恶性肿瘤相关的许多特征,包括细胞毒性治疗后的肿瘤复发,这是由于它们的增殖性休眠和由于在其分化期间获得的突变而允许产生抗性肿瘤细胞亚克隆的能力。在这里,我们证明使用极限稀释分析(ELDA),可以测量PI3K-突变致癌NSCs和非致癌NSCs之间的球形成潜力频率的细微差异体外。我们进一步展示了ELDA如何在强制分化之前和之后用于细胞,以放大突变体和对照NSCs之间的球形成潜力的固有差异。最终,ELDA利用单个或少数种子干细胞自我更新,分裂和形成神经球的能力差异。重要的是,该测定还允许在不同条件下在遗传上不同的细胞之间或相同细胞之间进行比较,其中可以测试靶特异性药物或其他新型癌症干细胞疗法的影响。
【背景】胶质母细胞瘤(GBM)是最常见的脑癌之一,预后极差(Kaye和Morokoff,2014)。 ...
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Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells
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Author:
Date:
2018-08-20
[Abstract] Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 μm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer’s group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in ...
[摘要] 纤连蛋白(FN)是一种细胞外基质蛋白,由许多细胞类型分泌,主要与细胞表面受体整合素α5β1结合。整合素α5β1结合启动FN逐步组装成原纤维,这一过程称为原纤维形成。我们和其他几个人已经证明了原纤维形成对细胞迁移和转移的关键作用。虽然免疫染色和显微镜方法有助于可视化FN掺入原纤维,每个原纤维的长度至少为3μm,但是第一项研究开发了一种生物化学分离FN以量化原纤维并入FN的方法,由Jean Schwarzbauer小组于1996年出版。我们的方案改编自原始出版物,并已在多种细胞类型上进行测试,包括如此处所示的MCF10A乳腺上皮细胞和Caki-1肾癌上皮细胞。使用两种洗涤剂提取物,将细胞FN分离成不溶于洗涤剂或掺入原纤维的FN和可溶性FN或未掺入的级分。为了确定原纤维形成是否利用FN的再循环池,我们使用了生物素标记的FN(FN-生物素)再循环测定,其已经从先前的研究中修改。使用再循环测定和脱氧胆酸盐分离方法的组合,可以定量地证明在不同实验条件下细胞中原纤维形成的程度,并确定原纤维形成的FN来源
【背景】 纤连蛋白(FN)是普遍产生的细胞外基质(ECM)组分(Uitto et al。,1989; Mao和Schwarzbauer,2005)。纤连蛋白库是转录产生的,可以通过几种生长因子如TGF-β1增加(Yokoi et al。,2002; Mimura ...
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