Name: isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)
Uploaded: 2013-01-14T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

我们感谢他们的帮助，流式细胞仪分选博士Yitzchak Oschry和博士ORIT的鹭阿西夫。这项研究得到了补助NE从欧盟第七框架计划（FP7/2007-2013）根据赠款协议Ň°[276890]，从以色列癌症协会（20110078），并从以色列癌症研究基金（ 研究职业发展奖 ）。

1。解剖小鼠乳腺皮肤组织<ol><li>解剖组织的小鼠之前，需准备以下用品及试剂： 耗材： <ul><li>外科工具（在用洗涤剂和洗涤，然后浸渍在70％的乙醇）。 </li><li>鼠标停留在剥离时的发泡胶面。 </li><li>引脚或针头在解剖小鼠。 </li><li>玻璃瓶盖的消化，含有磁性搅拌棒（灭菌）。 </li><li>水浴中于37℃，含有潜式磁力搅拌器和足够的水来淹没消化罐，休息时的搅拌盘上。 </li></ul><...

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虽然在组织培养中进行的实验可以是信息性 ​​的和建议可以验证在体内的功能原理，它是已知的，会发生大的变化，在基因表达的细胞在培养11,12。为了避免组织培养一步成纤维细胞中的基因表达分析时，我们开发了一个协议，允许隔离正常，以及与癌症相关的新鲜小鼠成纤维细胞或人体组织。该协议已成功应用于鼠标，从9人的皮肤，胰腺，乳腺组织。用流式细胞仪分?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;的试剂名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;<td<strong评论（可选）</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt; Gibco公司</td&gt;<td&gt; 41965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt;生物产业</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;Ⅱ型胶原酶</td&gt;<td&gt;沃辛顿</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;Ⅳ型胶原酶</td&gt;<td&gt;沃辛顿</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;脱氧核糖核酸</td&gt;<td&gt;沃辛顿</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;细胞过滤70微米</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td纯化的抗小鼠CD16/CD32</td&gt;<td&gt; BD Pharmingen公司</td&gt;<td&gt; 553142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;通过探测（7AAD）</td&gt;<td&gt; E-生物科学</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; ANTI-MOUSE的CD140a - PE（PDGFRA）</td&gt;<td&gt; E-生物科学</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;抗鼠F4/80- FITC</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM W / O酚红</td&gt;<td&gt; Gibco公司</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; I型胶原</td&gt;<td&gt; BD Biosciences公司</td&gt;<td&gt; 354236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

癌症相关的成纤维细胞（CAFS）是最重要的细胞类型，许多癌症的肿瘤间质内，特别是乳腺癌，其突出的存在往往与预后不良1,2。 CAF的活化的基质成纤维细胞的亚群，其中许多表达的肌纤维母细胞标记物α-SMA的3。 CAF的起源来自局部组织的成纤维细胞，以及从招募到显影的肿瘤和采用的CAF表型的影响下的肿瘤微环境4的骨髓来源的细胞。 CAF的信号，促进肿瘤细胞增殖，血管生成和侵袭5-8，促进肿瘤的发生，生长和发展。我们表明，CAF的提高肿瘤的生长，介导肿瘤促进炎症开始，最早在癌前阶段9。尽管越来越多的证据中发挥的关键作用CAF的促进肿瘤的生长，研究CAF的一直是一个持续的挑战，由于缺乏CAF-这些细胞的特异性标志物和广阔的异质性，与现有的许多亚型肿瘤微环境的10。此外，成纤维细胞的体外研究是通过阻碍它们的基因表达中的事实，常常在组织培养11,12改变。为了解决这个问题，让公正的新鲜小鼠的成纤维细胞和人体组织的基因表达分析，荧光激活细胞分选（FACS）13,14以前的协议的基础上，我们开发了一种方法。我们的方法依赖于利用PDGFRα隔离成纤维细胞的表面标记，从新鲜小鼠和人体组织。 PDGFRα中大量表达的正常成纤维细胞和纤维母9,15。此方法允许隔离纯种群的正常成纤维细胞和CAF的，包括，但不限于α-SMA +活化肌成纤维细胞。然后可以隔离成纤维细胞用于表征和比较基因的表达，在肿瘤发生在纤维母的演变。事实上，我们和其他人表达谱成纤维细胞分离细胞分选16。该协议成功地进行了高度浓缩的人群皮肤，乳腺，胰腺和肺组织中的成纤维细胞进行隔离和剖析。此外，我们的方法允许排序的细胞培养，在为了执行功能的实验，以避免污染的肿瘤细胞，这往往是一个很大的障碍时，试图来培养的CAF。

使用PDGFRα隔离高度富集的组织的成纤维细胞的种群中的成纤维细胞的结果作为一个标记。排序后的纯度为99％的水平，量化排序后分析（ 图2A）。估算污染的非成纤维细胞通过小区固有控制基因的相对表达量（ 图2B）的百分比通常显示0.1-0.6％的污染。这个级别的纯度，使高品质的转录组分析孤立的成纤维细胞9。 在乳腺中，在组织中的成纤维...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

癌相关成纤维细胞（CAFS）信号，促进细胞增殖，血管生成和炎症促进肿瘤的发生，生长和发展。在这里，我们描述了一种方法，分离出纯净的新鲜小鼠和人体组织的正常成纤维细胞和纤维母细胞分选，表面标志使用PDGFRα的人口。

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Research

JoVE Journal

Biology

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

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Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

科研

生物学

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The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

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溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

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Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

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After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

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Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

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During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

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荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...