JSO支持由美国国立卫生研究院露丝，属Kirschstein NRSA（F32 DK091040），AK是来自美国糖尿病协会（7-10-MI-05）的博士后奖学金支持，AHH支持由美国心脏协会成立研究者奖（12EIA8270000）。流式细胞仪检测实验进行了流式细胞仪在VMC共享资源。 VMC流式细胞仪共享资源支持范德比尔特消化疾病研究中心（DK058404）。

1。试剂和耗材发起这个实验协议之前，需准备以下试剂： <ol><li> 70％乙醇</li><li> 1X PBS </li><li> 1X的DPBS（无钙，镁）与0.5％牛血清白蛋白补充</li><li> FACS缓冲液：1X DPBS（无钙，镁），2mM的EDTA，1％FCS的</li><li> ACK缓冲液：150mM的氯化铵的10mM KHCO 3，和0.1mM EDTA在水中的Na 2 </li></ol> 2。脂肪组织的收获和准备<ol><li>安乐死老鼠IACUC批准程序，具体到每个机构。 </li><li>彻底弄湿皮草，用70％的乙醇。 </li><li>在剑突（胸骨下部）的水平做一个切口，打开胸腔，暴露心脏，小心不要断绝任何主要血管。 </li></ol> 注意：在这一点上，它也是有帮助的离开隔膜完好的一样多可能的，切一个缺口，肋骨的右侧，让血液和灌注液流出的胸腔。 <ol start="4"><li>剪辑的右心房，让血液灌流逃脱循环系统。 </li></ol> 注意：肥胖老鼠，多余的心包处理时可能需要拆除，允许进入心脏。 <ol start="5"><li>用钳子抓住心，轻轻插入一根针进入左心室通过顶点。慢慢灌注鼠标，用15毫升无菌PBS。 </li></ol> 注意：如果肺部开始，以填补和拓展，减少灌注率。 <ol start="6"><li>打开腹腔脂肪垫删除perigonadal的使用小心，以避免任何性腺组织。 </li><li>脂肪垫放置在称重船在冰含2毫升1X DPBS（无镁或钙）含0.5％BSA，AT成细片和肉。 </li></ol>注：限制量的每称重船1.2克的。如果量的超过1.2克时，均匀地分成两个称量皿之间。 <ol start="8"><li>查看AT样品在冰上，准备3毫升每AT样本组成的1X DPBS补充有0.5％BSA的10mM的CaCl 2和4毫克/毫升Ⅱ型胶原酶，胶原酶消化液。 </li></ol> 3。胶原酶消化<ol><li>传输到50毫升锥形管浇匀浆和漂洗1毫升DPBS（0.5％BSA）和3毫升Ⅱ型胶原酶消化液的权衡船。 </li><li> AT匀浆在旋转摇床（200转），在37℃孵育20分钟。 </li><li>添加10毫升DPBS（0.5％BSA）的锥形管置于冰上。 </li><li>磨碎匀浆无数次，使用10毫升血清吸管，并传递到一个新的50毫升锥形管细胞悬液通过100微米的过滤器。 </li><li>细胞悬浮液在500×g离心10分钟在4研发比如C。 </li><li>倒出上清，重悬细胞沉淀SVF ACK缓冲液3毫升溶解红细胞的污染。 </li><li>添加12毫升的FACS缓冲液中，离心细胞悬浮液在4℃下以500×g离心10分钟</li><li>倒出上清，重悬细胞沉淀SVF在流式细胞仪缓冲。 </li></ol> 注意：使用作为导览多少FACS缓冲液重悬英寸如果细胞沉淀覆盖的50毫升锥形管的底部，用FACS缓冲液0.5-1毫升，否则，在0.25〜0.5重悬细胞沉淀的大小毫升。 <ol start="9"><li>将样本放置在冰上混合加入40μlFACS缓冲液中，加入50μl台盼蓝溶液（0.2％），10微升细胞悬浮液制备1:10稀释每个样品的等分试样中，进行细胞计数。 </li><li>基于台盼蓝染色，计数活细胞，并稀释至终浓度为5-10×10 6细胞/ ml的细胞悬浮液。 </li></ol> 4。细胞表面抗原染色<o升&gt; <li>添加抗小鼠CD16/CD32抗体（Fc的块），以终浓度为0.5-1μg/106个细胞，并在冰上孵育10分钟。 </li><li>将样品（≥10 6个细胞），以12×75毫米的聚苯乙烯圆底管。准备单独的管，如果，分析取款机和T细胞，并准备了充足的管数以容纳所需的补偿和修饰的荧光减一（FMO）控制的额外的细胞结合。 </li></ol> 注意：作为一个例子，当量化的自动取款机的基础上，下面的补偿F4/80和CD11b及FMO控制的比例将需要准备： - 未染色（细胞） - DAPI或碘化丙啶（PI）单染色（细胞，不加可行性染料，直到步骤5.1） - ，F4/80的APC单染色（细胞或补偿珠） - 细胞CD11b FITC的单染色（细胞或补偿珠） - FMO 1（细胞）：大鼠IgG2a的κ型控制APC + CD11b的FITC +活力染料（在步骤5.1中添加） - FMO 2（细胞）：F4/80 APC +鼠IgG2b的κ型控制FITC +生存能力染料（增加5.1步） <ol start="3"><li>在相应的浓度添加的荧光基团共轭的第一抗体和/或同种型对照（ 见表试剂和材料 ）。 </li><li>保护样品免受光线和在4°C孵育30分钟。 </li><li>加入2毫升的FACS缓冲液中，离心细胞悬浮液在4℃下以500×g离心5分钟</li><li>倒出上清，重悬细胞沉淀SVF 2毫升FACS缓冲。 </li><li>细胞悬液500 XG离心5分钟，在4°C，重悬细胞沉淀SVF≥400微升FACS缓冲。 </li><li>将样品12×75毫米聚苯乙烯圆底配备35微米的细胞过滤管顶管。</li><li>保护光，店内样品在4℃，直到流式细胞仪分析。 </li></ol> 注意：为获得最佳效果，细胞应immeadiately分析，然而，与此协议的FACS分析已经成功地进行了对存储在FACS缓冲液中的标记的细胞在4℃下1-2小时如果细胞需要加以固定，以增加存储时间标记的细胞可以用2％多聚甲醛在4℃下固定24小时，FACS分析之前。抗体公司标记的细胞可被存储为一个星期，然而，这并没有此过程的范围内进行了测试。 5。流式细胞仪分析<ol><li>流式细胞仪分析前样品和适当的控制，增加生存能力染料，允许死/活细胞歧视。 </li></ol> 注意：众多的可行性染料是市售的，但DAPI和PI建议取决于激发/发射教授所使用的荧光团标记的抗体尔斯。 DAPI和碘化丙啶被添加到每个样品的终浓度为0.2毫克/毫升。 <ol start="2"><li>使用未染色的阴性对照样品，分离的细胞优选从相同的组织作为实验样品，进行调整使侧向散射光（SSC）和前向散射（FSC）的感兴趣的细胞群（次）的规模。 </li></ol> 请注意：有关的AT SVF细胞的分析，它是建议SSC被显示在对数标尺的线性标尺与FSC。 SSC使用日志规模分析是特别重要的，当自动柜员机（ATM），这往往是非常大的，颗粒状。 <ol start="3"><li>绘制一个初始光散射门类型细胞（s）被分析的基础上，调整增益，使光电倍增管（PMT）未染色的细胞是最左边的一个单参数直方图（约集中在10 2）相应的通道。&lt;/ LI&gt; </ol> 注意：这是建议的淋巴细胞和巨噬细胞（或其它髓系细胞）分别进行分析，由于自体荧光的差异。 <ol start="4"><li>使用单一的彩色控制或抗体捕获补偿珠进行多色彩补偿。 </li></ol> 注意：建议补偿有孔玻璃珠的使用，但是，每种抗体的兼容性必须得到保证。例如，补偿的有孔玻璃珠可能不交叉反应，兔抗体，在这种情况下，分离的细胞需要获得相应的单染色控制。 <ol start="5"><li>设置的基础上改进的FMO控制实验的大门。 </li><li>设置流式细胞仪收集相应数量的患病人口的利益基础上的事件，并记录实验数据。 </li><li>出口FCS数据文件进行离线分析。有许多方案可供流量cytometr的y数据分析。我们建议Cytobank，一个基于网络的平台，允许的investigatores存储和分析数据，并生成数字从任何一台计算机与互联网接入18。此外，Cytobank提供的能力，使数据的公共访问或限制访问的合作者。 </li></ol>

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我们什么都没有透露。

流式细胞仪的广泛使用，表征免疫细胞的AT导致肥胖的代谢结果，在免疫系统中的作用越来越大的兴趣。虽然确切的协议会有所不同，根据自己的经验和现有设备的实验室之间的关键步骤包括：胶原酶消化，差速离心和细胞表面抗原的标记。本文的目标是提供一个详细的协议和实用指南隔离AT SVF有限的工作经验，与AT和/或执行流式细胞仪调查。 任何实验技术，有无数的变化，...

从历史上看，脂肪组织（AT）一直被看作是惰性的脂质储存器官，膨胀和收缩响应能量平衡。我们现在明白了，在代表一个动态的内分泌器官，积极分泌一些激素，它直接影响摄食行为和全身葡萄糖稳态。此外，在过去十年中一直为众多的人口居住在血管基质部分（SVF），以及他们的贡献动态平衡免疫细胞的不断升值。 其次通过差速离心分离脂肪细胞和SVF使用胶原酶消化能力罗德贝尔首次描述了在1964年1。 Ⅱ型胶原酶是最常用的为脂肪细胞的和SVF分离的脂肪细胞的胰岛素受体​​1的维护。在早期，主要采用酶分馏AT学习体重减轻，脂肪CYTE代谢和隔离脂肪前体细胞。最近，这种技术，结合流式细胞仪和广泛的可用性市售荧光标记的抗体的数量不断增加，促进免疫细胞的表征AT。 虽然存在前面所述的免疫细胞在发炎AT 2，Weisberg的等人的开创性论文。徐等人 。发表于2003年首次记录积累的巨噬细胞（自动取款机）肥胖，分泌炎性细胞因子，并与AT特异性和全身胰岛素抵抗3,4。这些观测服务为基础，最近创造了一个新的研究领域，“immunometabolism，”5，并已跟进研究，涉及各种免疫细胞的人口，包括树突状细胞，肥大细胞，T细胞8 -10，B细胞，NKT细胞，嗜酸性粒细胞13，与肥胖相关的胰岛素抵抗的发展中的嗜中性粒细胞14,15。 这篇文章的目标是提供胶原酶消化技术，用于分离细胞的AT SVF和表征自动取款机和AT＆T的细胞通过流式细胞仪的详细说明。该协议已优化的鼠标，但是，观众可能会受益于阅读的优秀文章，优化这一技术为人类提供丰富的细节在16。这篇文章的目标受众包括有限的工作经验，用鼠标进行流式细胞仪的调查。平衡的时间和资源的细胞产率以及活性与几个实际的考虑，以及最佳的流式细胞仪控制AT的免疫细胞群的特征。除了我们的通讯协议，读者请参照最近的朱庇特的文章Basu 等的精彩讨论一些流式细胞仪技术方面的，包括适当的控制和补偿17。

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS (no Ca or Mg) 10 x 500 ml</td>
 <td>Life Technologies</td>
 <td>14190-250</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Life Technologies</td>
 <td>D3571</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A2153-100G</td>
 <td></td>
 </tr>
 <tr>
 <td>collagenase</td>
 <td>Sigma</td>
 <td>C6885-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>propidium iodide solution</td>
 <td>Sigma</td>
 <td>P4864-10 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 20 microliter</td>
 <td>Rainin</td>
 <td>SS-L10</td>
 <td></td>
 </tr>
 <tr>
 <td>20 microliter filter tips</td>
 <td>Rainin</td>
 <td>SRL10F</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 250 microliter tips</td>
 <td>Rainin</td>
 <td>SS-L250</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter tips</td>
 <td>Rainin</td>
 <td>GPS-L1000</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter filter tips</td>
 <td>Rainin</td>
 <td>GP-L1000F</td>
 <td></td>
 </tr>
 <tr>
 <td>250 microliter Filter Tips</td>
 <td>Rainin</td>
 <td>SR-L200F</td>
 <td></td>
 </tr>
 <tr>
 <td>FC Block</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td></td>
 </tr>
 <tr>
 <td>fisher 100mn strainers</td>
 <td>Fisherbrand</td>
 <td>22-363-549</td>
 <td></td>
 </tr>
 <tr>
 <td>medium weigh dish</td>
 <td>Fisherbrand</td>
 <td>02-202B</td>
 <td></td>
 </tr>
 <tr>
 <td>aluminum foil</td>
 <td>Fisherbrand</td>
 <td>1213100</td>
 <td></td>
 </tr>
 <tr>
 <td>mincing scissors</td>
 <td>Fisherbrand</td>
 <td>089531B</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisherbrand</td>
 <td>2215365</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>14-959-49A</td>
 <td></td>
 </tr>
 <tr>
 <td>filter top FACS tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml pipette case 200</td>
 <td>BD Falcon</td>
 <td>1367520</td>
 <td></td>
 </tr>
 <tr>
 <td>round bottom tubes</td>
 <td>BD Falcon</td>
 <td>352058</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml syringe</td>
 <td>BD Falcon</td>
 <td>309646</td>
 <td></td>
 </tr>
 <tr>
 <td>V Bottom Plates</td>
 <td>Costar</td>
 <td>07-200-107</td>
 <td></td>
 </tr>
 <tr>
 <td>transfer bulb pipette</td>
 <td>Thermo Scientific</td>
 <td>13-711-22</td>
 <td></td>
 </tr>
 <tr>
 <td>Shaker</td>
 <td>Thermo Scientific</td>
 <td>11 676 071</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive mat</td>
 <td>Thermo Scientific</td>
 <td>1368750</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Culture Centrifuge</td>
 <td>Sorvall</td>
 <td>75253839</td>
 <td></td>
 </tr>
 <tr>
 <td>Adapters</td>
 <td>Sorvall</td>
 <td>75003723</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD16/CD32</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Concentration: 0.5 - 1 &mu;g/ 106 cells</td>
 </tr>
 <tr>
 <td>Rat anti-mouse F4/80</td>
 <td>eBioscience</td>
 <td>17-4801</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 17-4321</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD11b</td>
 <td>eBioscience</td>
 <td>11-0112</td>
 <td>Fluorophore conjugate: FITC 
 Concentration: 0.5 &mu;g/ 106 cells 
 Isotype control catalog number: 11/1/4031</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse CD11c</td>
 <td>eBioscience</td>
 <td>12-0114</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: Dec-88</td>
 </tr>
 <tr>
 <td>Goat anti-mouse MGL1/2</td>
 <td>R&amp;D Systems</td>
 <td>FAB4297P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Goat anti-mouse CD206</td>
 <td>R&amp;D Systems</td>
 <td>FAB2535P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CCR2</td>
 <td>R&amp;D Systems</td>
 <td>FAB5538P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC013P</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse TCR&beta;</td>
 <td>BD Biosciences</td>
 <td>553174</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 553956</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>552877</td>
 <td>Fluorophore conjugate: PE-Cy7 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: 552784</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD4</td>
 <td>BD Biosciences</td>
 <td>557956</td>
 <td>Fluorophore conjugate: Alexa Fluor 700 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 557963</td>
 </tr>
 </tbody>
</table>
Table 1. List of Materials and Reagents.

isolation of adipose tissue immune cells

在脂肪组织中的巨噬细胞浸润增加（AT）的肥胖啮齿类动物和人类的发现感兴趣的免疫细胞贡献的加剧，导致局部和全身的胰岛素抵抗。瘦肉和肥胖不同的免疫细胞群的分离和量化是现在普遍使用的技术，还必须格外小心，应采取使血管基质细胞的分离和流式细胞仪分析获得的数据是可靠的，可解释的在immunometabolism实验室。在这段视频中，我们展示了如何切末，消化，并隔离富含免疫细胞间质血管分数。随后，我们将展示如何抗体标记巨噬细胞和T淋巴细胞，以及如何正确的门对他们在流式细胞仪实验。代表流式细胞仪地块从低脂肪喂养的瘦和高脂肪喂养的肥胖小鼠。在此分析中的一个关键因素是使用的抗体，该抗体荧光渠道在巨噬细胞是自然自发荧光，以及使用适当的补偿控制。

雄性小鼠附睾脂肪垫的AT胶原酶消化后差速离心法分离的SVF喂食低脂肪（10％千卡来自脂肪）或高脂肪（60％千卡来自脂肪）饮食（LFD和HFD ），分别为16周。然后SVF细胞标记的荧光团标记的第一抗体，通过FACS分析可行的自动取款机（ 图1）和AT＆T的细胞（ 图2）的量化比例。 图1A和2A中描绘了初始的门控，包括光散射，双重歧视和生存能力门。应该?...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Immune Cells at JoVE.com

Isolation of Adipose Tissue Immune Cells

脂肪组织（AT）是一个强烈的免疫细胞的激活和相互作用的部位。几乎所有的免疫系统的细胞是存在于AT和它们的比率是改变了肥胖。适当的隔离，定量，定性的AT免疫细胞群，是了解自己的角色在immunometabolic疾病的关键。

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Research

JoVE Journal

Immunology and Infection

35.9K 次观看.  Vanderbilt University School of Medicine. 脂肪组织（AT）是一个强烈的免疫细胞的激活和相互作用的部位。几乎所有的免疫系统的细胞是存在于AT和它们的比率是改变了肥胖。适当的隔离，定量，定性的AT免疫细胞群，是了解自己的角色在immunometabolic疾病的关键。

视频: Isolation of Adipose Tissue Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

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

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

肾脏免疫细胞的可靠和高效率的提取

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...