Name: isolation of mouse peritoneal cavity cells
Uploaded: 2010-01-28T16:45:00
Duration: 4 min 32 s
Description: Watch this Scientific Journal Video about Isolation of Mouse Peritoneal Cavity Cells at JoVE.com

这项工作是支持部分由美国国立卫生研究院授予AI069358和BloodCenter研究基金会。

<ol><li>开始之前的过程中，下列项目需要收集和准备： <ul><li>冰</li><li> 27克针5ml注射器</li><li> 25克针5ml注射器</li><li>安装鼠标的泡沫塑料块和引脚</li><li>可以放在托盘上安装块</li><li>剪刀和镊子</li><li>收集管</li><li> 70％的乙醇</li><li> PBS与3％小牛血清（FCS）（前冷冻保存在冰） </li></ul></li><li>安乐死的鼠标，喷以70％乙醇和安装在它的后面的发泡胶块。 </li><li>使用剪刀和镊子切开腹膜外的皮肤，轻轻一拉，内层衬里的腹腔暴露。 </li><li>使用27克针进入腹膜腔注入5毫升冰冷的PBS（含3％FCS）。慢慢推针腹膜穿刺小心，不要被任何机关。 </li><li>注射后，轻轻按摩腹膜驱逐任何贴壁细胞的PBS液。 </li><li>插入一个25克的针，锥，附着在腹膜5 ml注射器和搜集流体移动时针尖轻轻地避免脂肪组织或其他器官堵塞。尽可能收集尽可能多的液体和存款取出后从注射器针管放在冰上收集细胞悬液。 可选：重复步骤4-6 </li><li>请在腹膜内的皮肤切口，并与钳皮肤而使用塑料的巴斯德吸管收集剩余的液体从腔。 </li><li>如果在步骤6或7可见血液污染检测，然后被污染的样品应该被丢弃。 </li><li>收集的细胞悬液在1500转的旋转，8分钟，弃去上清液，并计算所需的媒体或PBS重悬细胞。 </li></ol>替代协议获得硫乙醇酸盐引起巨噬细胞： 使用此方法，以获得更高的巨噬细胞的产量。 <ol><li> 3％（W / V）布鲁尔硫乙醇酸盐培养基（7）到每只小鼠腹腔注射5毫升。 </li><li>等待3-5天，并继续执行步骤1段收集细胞。 </li></ol> nonelicited方法相比，可以收集到约10倍多的巨噬细胞。唯一担心的是，此过程中获得的巨噬细胞，在他们的一些生理特性不同。 代表性的成果： 从unmanipulated鼠标，5-10万元的腹腔细胞，可以得到具有良好的隔离协议。在所有活细胞，B细胞的50-60％，〜30％的巨噬细胞和5-10％的T细胞（图1）。 <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="图1" /> 图1。从腹膜腔中分离的细胞表型。以下隔离腹腔细胞，他们沾满抗鼠TCRβ- FITC，B220 - PE德州红，CD11b的太平洋蓝，CD23 - PE - Cy7和CD5 - APC。代表流式细胞仪图显示B细胞和T细胞（A），巨噬细胞（B），B1和B2的细胞（C）和阿维菌素B1a和B1B细胞（D）的百分比。

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腹腔细胞的分离，不同的免疫细胞，主要是巨噬细胞和特定的B细胞亚群的研究是一个重要的技术。虽然这是一个简单的过程，也有一些涉及的关键步骤。应避免污染所收集的样品中血液的存在，获得纯腹腔细胞群。颈椎脱位安乐死应做仔细，以避免在腹腔的血液污染。或者可以使用的CO 2。此外，在手术过程中适当的照顾中应采取，以避免在腹腔穿刺膀胱或任何其他机关。注入流体恢复大部分也是很重要的细胞产量。 此过程被广泛用于研究自中等数量的居民巨噬细胞生物学，未刺激巨噬细胞可以很容易地获得从腹腔，在髓系祖细胞在体外分化成成熟的巨噬细胞，巨噬细胞集落刺激因子（8费力的任务，9）。从腹腔巨噬细胞的产量可以提高使用硫乙醇酸盐启发式，虽然硫乙醇酸盐的使用可能会改变的巨噬细胞的生理特性（7）。 B细胞是先天和适应性免疫中扮演关键角色，我们的免疫系统的重要组成部分。在不同的B细胞亚群，B1细胞构成独特的先天免疫，自身免疫和免疫调节B细胞参与的一个子集。 B1细胞主要位于腹腔和自我补充。它们是自然的IgM它提供了针对病毒和细菌（10-12）保护的第一线的主要生产商之一。 B1细胞的IL - 10（6），这是一个重要的细胞因子参与免疫调节的主要来源。虽然几项研究已经与B1单元格的追求，仍然有余地为进一步评价其​​对比功能，特别是其监管作用。腹腔细胞的分离方法研究B1细胞的功能提供了独特的机会。

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isolation of mouse peritoneal cavity cells

腹膜腔是腹腔膜结合的充满液体的哺乳动物，其中包含的肝，脾，大部分的胃肠道和其他脏器。港口包括巨噬细胞，B细胞和T细胞的免疫细胞的数量。一个天真腹腔巨噬细胞大量存在，使一个天真的组织驻地巨噬细胞（1）收集的首选网站。腹膜腔是因为存在一个独特的腹腔居民的B细胞为B1细胞除了常规B2细胞的一个子集B细胞的研究也很重要。 B1细胞分为菌素和B1B细胞，它可以通过表面的CD11b和CD5表达尊敬。 B1单元格是一个由多种病原体（2-4）早期保护自然提供的IgM的重要来源。这些细胞是自身反应（5）性质，但它们是如何控制，以防止自身免疫性疾病仍然是不完全理解。相反，CD5<sup&gt; +</sup凭借他们的IL - 10的生产能力（6）&gt;阿维菌素B1a的细胞具有一定的调节特性。因此，腹腔B1细胞研究，因为他们的不同的功能和与他们的发展及规管相关的许多未解决的问题是一个有趣的细胞群。腹腔居民的免疫细胞的分离是非常棘手，因为缺乏一个定义的结构腹腔内。我们的协议，将描述一个获得可行的免疫细胞，从小鼠的腹腔，然后可以使用的表型分析，流式细胞仪和不同的生化和免疫学检测程序。

Watch this Scientific Journal Video about Isolation of Mouse Peritoneal Cavity Cells at JoVE.com

Isolation of Mouse Peritoneal Cavity Cells

在哺乳动物的腹腔中包含不同的免疫细胞群的先天免疫反应的关键。这些细胞的生化和功能分析的一个有效的隔离方法是必需的。在这里，我们提供一个全面的方法在小鼠腹腔细胞隔离。

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Research

JoVE Journal

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从大鼠睾丸支持细胞的分离和管周细胞

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Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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小鼠肾上腺的分离、固定和免疫荧光成像

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增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...