Name: murine dermal fibroblast isolation by facs
Uploaded: 2016-01-07T10:00:01
Description: Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

这项工作是由美国国立卫生研究院资助R01 GM087609（到HPL），由英格丽·莱和比尔舒纪念安东尼·舒（到HPL），美国国立卫生研究院资助U01 HL099776（以MTL）的礼品，在Hagey实验室的资助部分支持小儿再生医学和橡树基金会（至MTL，GCG和HPL）。 GGW是由斯坦福大学医学院，斯坦福大学医学院的科学家培训项目的支持，并NIGMS培训资助GM07365。 ZNM是由整形外科基金会研究奖学金资助和Hagey家族基金的支持。 MSH是由美国加州再生医学研究所（CIRM）临床研究员的培训补助TG2-01159，颌面外科医生协会（ASMS）/颌面外科基金会（MSF）研究基金奖，并在移植与组织工程研究金支持。

该协议遵循经对实验动物的斯坦福大学行政专家组的方法。 1.消化鼠真皮<ol><li>安乐死小鼠麻醉后颈椎脱位与腹膜内注射氯胺酮100mg / kg的甲苯噻嗪+ 20mg / kg的+乙酰丙嗪3毫克/公斤。 注意：各种年龄和背景都可以使用。 </li><li>刮脸和脱毛的背部皮肤。大约100,000细胞可以从一块背部皮肤的60毫米×100毫米的分离。 </li><li>淹没鼠标在70％的乙醇和地点上的无尘，无菌的表面干...

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在这个手稿中描述的协议提供了通过基于FACS分选来分离成纤维细胞，在比较现有方法，这两种选择的亚群或随后的分析之前需要时间在细胞培养物的方法。从收获的皮肤所需的成纤维细胞的分选的时间是约6小时;然而，在收获用于小鼠的数目将影响这一估计。 有几个点在协议中需要特别的照顾。第一是由以下中的分离过程的消化和离心除去脂肪消化和除去上清液的上部脂质?...

成纤维细胞经常定义形态作为坚持以塑料基板梭形细胞。成纤维细胞是负责合成和重塑细胞外基质在胚胎和成年器官1中的原理的细胞类型。成纤维细胞因此至关重要哺乳动物发育和细胞外环境是影响相邻存在于各组织和器官中的细胞类型的行为有助于基本上。 成纤维细胞也落后造成巨大的临床负担的医疗条件多样化的主要细胞类型。病理的成纤维细胞活性的损害正常组织功能，并且包括组织和器官的纤维化（如肺和肝），疤痕下列皮肤伤口愈合，动脉粥样硬化，全身性硬化症，和形成血管损伤后2-5粥样硬化斑块的。特别是伤口愈合，急性和慢性，涉及ðeposition既不酷似也不功能类似正常组织周围，并导致发病率显著跨不同病理状态的疤痕组织。损伤后，存在成纤维细胞的肌成纤维细胞，以过渡，然后分泌结构ECM成分，发挥对邻近的细胞类型旁分泌效果，并且通过沉积疤痕组织6恢复机械稳定性。 在皮肤组织存在于跨越发育时间伤口修复的质量解剖部位之间和显著变化。在人生的前两三个月的胎儿愈合，不留疤痕;然而，从和整个成年期的晚期，治愈人类与疤痕。位点特异性，除了年龄特异性，在伤口愈合的差异存在。在口腔中的伤口改造以最小的疤痕形成7,8-，而皮肤伤口内的瘢痕组织沉积是显著9。争议仍然存在Çoncerning环境与对伤口愈合的结果本地的成纤维细胞的固有性质的在问候年龄和位置10,11的相对影响。定在小鼠口服与皮肤真皮和早期胚胎（E15）愈合的显著差异与购买胚胎（E18）真皮，它很可能是在某些发育年龄和各种解剖部位之间在成纤维细胞的种群特性存在差异。 1986年，哈罗德·F.德沃夏克假定肿瘤伤口不愈合12。 Dvorak的结论是，肿瘤行为类似于伤口在主体和通过激活伤口愈合的主机的响应诱导其基质。许多研究以来研究了成纤维细胞，以癌13-15的进展的贡献，但如在伤口愈合中，身份并有助于皮肤CA的基质隔室的成纤维细胞的胚胎起源的情况下rcinomas还没有得到充分的界定。在回答这个问题负有鉴于最近的研究暴露肿瘤相关成纤维细胞作为抗癌治疗16潜在有效靶点医疗相关性。 识别和前瞻性隔离赋有纤维化潜力体内成纤维细胞系是实现有效地操纵其范围广泛的急性和慢性疾病损伤反应的一个重要步骤。在1987年，科马克表明两个亚群的成纤维细胞，一种驻留在乳头和一个网状真皮层17,18内内。第三个亚群被发现在毛囊19,20的毛乳头区毛囊有关。当进行培养，成长潜力，形态和生长因子/细胞因子，这些成纤维细胞亚型表现出不同型材21-24。 迄今为止，研究探讨成纤维细胞，他terogeneity在很大程度上未能充分表征体内成纤维细胞之间的发育和功能的多样性。这部分地是对培养的成纤维细胞种群的依赖和细胞培养物或阳性选择的不是由所有的成纤维细胞25表示的自表面受体的基础上的均化效果的结果。从我们的实验室最近的一项研究证实了深刻的表面标志，并在培养与未培养成纤维细胞中分离的流式细胞仪为基础的分离方法，在此手稿26呈现转录变化。 接着，我们确定了鼠背真皮内的特定的成纤维细胞谱系并确定此谱系，通过ENGRAILED-1的胚胎表达的定义，主要负责在背部皮肤结缔组织沉积。期间急性和慢性纤维化形式，包括伤口愈合，癌症间质的形成，和谱系的功能辐射诱发的纤维化27。鲜明的成纤维细胞系的表征具有治疗旨在调节纤维化的行为至关重要的意义。 而不是使用依赖在体外操作 ，以实现细胞分离28,29的现有协议，收获协议（图1）这里详述将有助于成纤维细胞更准确地捕捉和表型的行为的体内产率信息进行分析。

<table border="0" cellpadding="0" cellspacing="0" width="1000">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">Surgical Forceps</td>
			<td style="width:369px;">Kent Scientific</td>
			<td style="width:108px;">INS650916</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elastase</td>
			<td>Abcam</td>
			<td>ab95133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>A14430-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium-Chloride-Potassium (ACK) lysing buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD31</td>
			<td>BioLegend</td>
			<td>102421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45</td>
			<td>BioLegend</td>
			<td>103125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tie2</td>
			<td>BioLegend</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ter-119</td>
			<td>BioLegend</td>
			<td>116233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EpCAM (CD326)</td>
			<td>eBioscience</td>
			<td>48-5791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">propidium iodide (PI viability stain)</td>
			<td>BioLegend</td>
			<td>421301</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine dermal fibroblast isolation by facs

成纤维细胞是负责分泌细胞外基质的原则细胞类型和许多器官和组织的重要组成部分。成纤维细胞的生理和病理背后临床实体，包括的纤维化在多个器官，增生性疤痕以下烧伤，缺血心脏功能的损失，和癌症间质的形成的频谱。然而，成纤维细胞仍然是一个不良的特征类型的细胞，主要是由于其固有的异质性。成纤维细胞的分离现有方法需要的时间在细胞培养的深刻影响细胞的表型和行为。因此，许多研究调查的成纤维细胞生物学依靠在体外操作 ，不准确捕获体内成纤维细胞的行为。为了克服这个问题，我们开发了一种基于FACS的协议，用于成纤维细胞的成年小鼠的背部皮肤，不需要细胞培养物中分离，由此preservi纳克生理转录和每个细胞的蛋白质组曲线。我们的策略允许通过一个谱系负栅极排斥非间充质细胞系（林- ），而不是一个积极的选择策略，以避免预选的成纤维细胞亚群的或富集表达特异性表面标志，并尽可能具有包容性跨越这个异类细胞类型。

这种方法（图1）的有效性已经在许多方面，它可以详细检查在我们最近的出版物27中得到验证。这些措施包括分选的细胞和细胞的大量和新鲜排序细胞的单细胞转录分析的免疫细胞化学。成纤维细胞进行排序，而不是直接更准确地依赖于文化的捕捉它们在体内的表型。使用谱系阴性耗竭的方法（图2A），而不是阳性选择方法避免预选特定亚群。这种方法的值由Rinkevich 等...

Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

Murine Dermal Fibroblast Isolation by FACS

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Research

JoVE Journal

Developmental Biology

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

科研

发育生物学

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

小鼠输卵管的显微解剖和解离：个体片段鉴定和单细胞分离

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

从未受干扰和受伤的小鼠骨骼肌中分离和培养纤维脂肪祖细胞和肌肉干细胞的FACS-分离和培养

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Cistrome Analysis in Mouse Muscle Stem Cells (Video) | JoVE

一种用于FACS分离的小鼠卫星细胞的CUT&amp;RUN分析的有效方案

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...