我们感谢他们在拍摄和视频制作的关键作用迪克弗利和Leigh金博尔。

1。外周血单个核细胞（PBMC）隔离<ol><li>获取血液样本 - 血液应收集在注射器或血管和（1:50的比例）与肝素的抗凝固，防止凝血。预计产量约1 × 10 6外周血单个核细胞每毫升血液-其中约40％将CD4阳性（CD4 +）T细胞。 </li><li>分装成50 ml锥形管，每管25毫升的血液。如果血液分离，轻轻混匀分发血浆前aliquoting </li><li>添加PBS中，使总体积至40毫升和11毫升的聚蔗糖底图，进入血液管插入，并仔细清除吸管吸管援助。聚蔗糖会慢慢流入试管底部。一旦有均衡的水平，慢慢取出吸管从血液管和丢弃。 </li><li>第血液管和迁入Aerosolve罐。坚决关闭在900和离心罐× G 20分钟 - 没有刹车。至关重要的是，没有刹车是在本自旋月底施加制动力会扰乱细胞层。 </li><li>分拆后，外周血应该形成一个独特的一层白色，黄色的等离子（上）和透明的聚蔗糖（下同）之间。轻轻脱脂白血细胞层，使用移液管转移到新管。可能制订了一些等离子和聚蔗糖与外周血单核细胞，但要避免制订任何红细胞层。气血两管通常可以在这一步相结合，以增加颗粒大小到一个单元格管。 </li><li> PBS中，使总体积至50 mL和离心机在500 × 15分钟，克 - aerosolve罐低制动。 </li><li>使用巴斯德吸管吸液，注意不要扰乱沉淀</li><li>治疗溶血性缓冲，每个试管中加入5-6毫升，轻轻混匀删除团块。孵育不超过5分钟。 </li><li> 10分钟，加入PBS中，总量将在230至50 mL和离心机× G - 低制动。 Aerosolve罐不再需要这些较慢的旋转。 </li><li>洗两次:使用巴斯德吸管吸液，填补管在230 × g 10分钟，用PBS和离心机。 </li><li>到最后的洗涤步骤之前，请删除重新悬浮细胞等份，台盼蓝稀释，和计数使用血球。这种细胞计数，将决定在T细胞的分离步骤所用试剂卷。 </li></ol> 2。 CD4 + T细胞分离* <ol><li>吸液和添加运行缓冲液，使总量高达10亿个细胞的40％微升。这通常需要30 UL的缓冲加上剩余的沉淀量。 </li><li>本卷转移到15 mL锥形管。 </li><li>在冰上10分钟添加CD4提取试剂盒 - 抗体鸡尾酒10 UL每10万个单元格，帽筒，并将其放置</li><li>管取出冰，删除帽，并添加30 UL运行，每10万个单元格的缓冲区</li><li>添加CD4提取试剂盒磁珠 - 20 UL每10万个单元格，帽筒，并置于冰上15分钟</li><li>新增10卷运行缓冲液洗，在230 × g离心10分钟。 </li><li>在旋转，设置在工作区中的磁铁和5 mL聚丙烯管</li><li>吸液体从细胞沉淀，加入2 ml运行缓冲和删除使用移液管的团块</li><li>使用相同的移液管，磁铁细胞转移，15分钟到5毫升的管和孵育</li><li>小心倒出的CD4 +细胞的一小部分成为一个显着的15毫升管反相磁铁和排空所有，但最后一滴血</li><li>运行缓冲液5毫升管的另一个2 mL的添加和删除从磁铁</li><li>轻轻冲洗掉管转移到一个显着的15 mL试管双方的CD4细胞</li><li>每管加入2 ml T细胞培养基，细胞计数和离心10分钟，分装在230 × g删除。 </li><li>细胞分装和稀释台盼蓝计数使用一个血球。这些细胞计数将决定用于扩展文化一步井的数量。 </li></ol> *另外，MACS列珠（美天旎生物技术公司），AutoMACS细胞分离机（美天旎生物技术公司），或Robosep细胞分离机（干细胞技术），可根据使用到生产指令代替步骤2.2 - 2.11。 3。体外扩增培养<ol><li>吸液的CD4 +和CD4细胞颗粒，根据细胞计数，文化传媒（通常为3万的CD4 +细胞每毫升10万每毫升的CD4细胞工程以及设立文化）。扩展文化需要3-5万CD4细胞，每孔2-3万CD4 +在总体积为1 mL培养基，在48孔培养板，每孔细胞。通常情况下，CD4 +细胞是限制人口</li><li>如果需要，照射的CD4细胞（5000拉德<html> S）。分装成井的适当数量的CD4细胞在48孔板中加入300-500微升到每个。板放置在37℃培养箱中培养1小时。贴壁CD4细胞作为抗原提呈细胞在扩张的文化。 </li><li>从培养箱取出钢板。 </li><li>加入500 uL的新媒体，轻轻吹打向上和向下转移吸管使用12-20倍，并删除所有的液体洗井。这洗会留下一层附着的抗原提呈细胞。 </li><li>当清洗完成后，添加刚够媒体湿每口井的底部（约100 UL） - 否则，贴壁细胞，将干出来的</li><li>新增2-3万CD4 +细胞，每孔。如有必要，添加额外的媒体带来的总量在每口井最多至1 mL。 </li><li>每口井所需的肽。为刺激经济增长的典型浓度是10微克/毫升的最后。 </li><li>板放置在37℃培养箱中培养7天。 </li><li>经过7天，加50 UL的Hemogen IL - 2（或同等学历，如重组IL - 2，10国际单位/毫升），每孔。 </li><li>其后每一天，用显微镜监测细胞生长。饲料尚未卸下一半的媒体融合，加入新鲜的媒体和50 UL的IL - 2井。分裂resuspending的细胞，细胞的一半移动到一个空井，加入新鲜的媒体和50 UL的IL - 2融合井。返回到孵化器的细胞。 </li><li>不断扩大的细胞，将准备为四聚体后13-15天文化。 </li></ol> 4。可视化四聚体染色的T细胞<ol><li>购买或组装四聚体相匹配的肽/ MHC的组合匹配的刺激CD4 + T细胞（见四聚体试剂的来源的讨论）。四聚体应〜0.5毫克/毫升的解决方案。 </li><li>删除板从孵化器</li><li>小心吸取了媒体的一半，从每口井</li><li>等分从每孔细胞 - 转移到一个5毫升的聚苯乙烯流式细胞仪管 - 通常为75μL或约50,000个单元格</li><li>四聚体 - 通常每50μL总体积1μL - 并在37个地点° C培养箱中培养1-2小时。这也是最好的一个不匹配的四聚体作为阴性对照的细胞染色从每口井的第二等份。 </li><li>标签细胞抗CD3，抗CD4，并通过添加3-10μL每个抗体的抗CD25抗体。可用于额外或替代的抗体标记。然而，不使用PE标记的抗体，因为这个通道必须保留的四聚体。此时，标签也单一颜色或同型控制使用多余的细胞。 </li><li>孵育抗体标记的细胞为15-30分钟在冰上或4 ° C。 </li><li>与运行在230 × g 10分钟的缓冲和离心机0.5至2毫升每管清洗。 </li><li>小心倒出的液体从流式细胞仪管。商店在随后的流式细胞仪分析（最好是在冰）为有盖容器内所有的管子。 </li></ol> 5。流式细胞仪的采集和分析<ol><li>校准流式细胞仪使用参考珠。 </li><li>验证仪器设置: <ol><li>使用未染色的对照组细胞或同型对照，调整位置，可行的淋巴细胞门（FSC与SSC），。围绕每个轴的第一个十年以下的人口（通过改变增益设置），从所有渠道中删除的自体荧光。 </li><li>使用单一的颜色控制管，每个单一颜色的正确象限内的中心事件（通过改变补偿设置）。 </li><li>做微调的仪器设置，使用与一个毫不相干的四聚体染色的管。在特别设置的PE通道可能需要调整，往往因为四聚体染色的抗体比控制更亮。不相干的四聚体染色&lt;0.5％的CD4 +细胞。 </li></ol></li><li>每管获取数据<ol><li>收集每个样品管和阴性对照管的事件</li><li>采集和保存至少20,000观活动</li></ol></li><li>分析数据<ol><li>导入的数据文件到流式细胞仪分析软件（如CellQuest软件，WinMDI，或FlowJo）。使用管使用一个不相关的四聚体染色，开始您的分析。 </li><li>剧情FSC与SSC和活的淋巴细胞（图1，左上面板）周围绘制一个门。 </li><li>剧情CD4 +与CD3 +（门控显示生活淋巴细胞）和借鉴盖茨周围的CD3 +和CD4 +人群（图1右上角面板）。 </li><li>情节的CD4与四聚体（门只显示CD3 +淋巴细胞）设置的象限边界的CD4 +四聚体+人口不到0.5％（图1底部左侧面板）。 </li><li>剧情+ CD25与四聚体（门控显示CD4 +淋巴细胞），使CD25 +四聚体以上人口是小于0.5％（图1右下角面板）设置的象限边界。 </li><li>分析没有改变的大门，所有样品管。 </li></ol></li></ol>1167/JOVE％20FIGURE％201.png"ALT ="图1"宽度="600"HEIGHT ="342"/&gt; 图1代表负控制四聚体染色结果。左上方面板显示着分散的诗句侧散射和淋巴细胞的大小门（R1）。右上角的面板门R1的显示与抗CD3和CD3 +（R）和CD4 +（R3）的门蚂蚁CD4。左下面板R2的门，并显示与四聚体的抗CD4。右下面板R3的门，表明抗CD25与四聚体。为四聚体地块的四个象限，分别设置为0.5％。 6。代表性的成果<ol><li>四聚体阳性细胞应扩大CD4 +细胞（图2）中的一个不同的人口。 </li><li>四聚体阳性细胞通常CD25 +调节性和经常的CD4高。 </li><li>在某些情况下，一个的MHC /肽结合，可以给背景染色（通常是由肽溶解性差造成）。从一个真正的积极的，因为"假阳性"细胞是不是一个单一的四聚体或CD25与四聚体图（图3）与CD4不同的人口，这样的背景下染色可以区别。 </li></ol><img src="/files/ftp_upload/1167/JOVE%20FIGURE%202.png" alt="" width="600" height="362" /> 图2代表积极的四聚体染色结果。面板的布局和闸门战略是一致的图1。作为一个独特的高人口与四聚体的抗CD25与四聚体面板的面板和鲜明的CD25 +人口的抗CD4 CD4四聚体阳性细胞出现。 <img src="/files/ftp_upload/1167/JOVE%20FIGURE%203.png" alt="图3" width="600" height="336" /> 图3代表"假阳性"四聚体染色结果。面板的布局和闸门战略是一致的图1。与四聚体面板的抗CD4显示一个模糊的"mounded"染色。抗CD25与四聚体也显示了一个"mounded"染色，没有明显的趋势，对CD25 +调节性。

<ol>
	<li>Novak, E. J., Liu, A. W., Nepom, G. T., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+Class+II+tetramers+permit+detection+and+characterization+of+peptide-specific+human+CD4++T+cells+proliferating+to+influenza+A+antigen.">MHC Class II tetramers permit detection and characterization of peptide-specific human CD4+ T cells proliferating to influenza A antigen.</a> J. Clin. Invest. 104, R63-R67 (1999).</li><li>Novak, E. J., Liu, A. W., Gebe, J. A., Falk, B., Nepom, G. T., Falk, B., Koelle, D. M., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetramer-guided+epitope+mapping:+rapid+identification+and+characterization+of+immunodominant+CD4++T+cell+epitopes+from+complex+antigens.">Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.</a> J. Immunol. 166, 6665-6670 (2001).</li></ol>

了解免疫的CD4 + T细胞的作用是极其重要的。然而，抗原特异性CD4 + T细胞可以用传统方法难以发现和隔离。相比之下，MHC II类四聚体，使所需的抗原特异性CD4 + T细胞的直接可视化。这个视频演示的分离，纯化，并在体外扩增的CD4 + T细胞，并随后将其可视化，使用四聚体。 II类四聚四是荧光蛋白偶联物的水溶性生物素化的II类的α/围绕一个荧光标记的链霉亲和的核心（图4）结合β二聚体组成。在本?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Class 2 Laminar flow biosafety cabinet</td>
<td>equipment</td>
<td>Thermo Forma</td>
<td>1200</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>50 mL conical Tube</td>
<td>equipment</td>
<td>VWR</td>
<td>47747-182</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>1X PBS (Ca/Mg free)</td>
<td>Reagent</td>
<td>HyClone</td>
<td>SH30028.02</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Ficoll-paque PLUS</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1440-03</td>
<td>foil wrapped to protect from light</td>
</tr>
<tr>
<td>Pipet Aid XP</td>
<td>equipment</td>
<td>Drummond</td>
<td>4-000-101</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>10 mL pipet</td>
<td>equipment</td>
<td>Corning/Costar</td>
<td>CLS4492</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Aerosolve Canisters</td>
<td>equipment</td>
<td>Beckman</td>
<td>359481</td>
<td>with 50 mL inserts</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>equipment</td>
<td>Beckman</td>
<td>GS-6R</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Transfer pipets </td>
<td>equipment</td>
<td>Samco</td>
<td>202-20S</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Pasteur pipets</td>
<td>equipment</td>
<td>VWR</td>
<td>14672-200</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Hemolytic Buffer</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>8.3 g/L NH4Cl, 1.0 g/L NaHCO3, 0.04 g/L disodium EDTA</td>
</tr>
<tr>
<td>Trypan blue</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6146</td>
<td>0.2% in PBS</td>
</tr>
<tr>
<td>Hemocytometer</td>
<td>equipment</td>
<td>VWR </td>
<td>15170-208</td>
<td>&nbsp;</td>
<tr>
<td>15 mL conical tube</td>
<td>equipment</td>
<td>VWR</td>
<td>05-527-90</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Running Buffer</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>PBS + 2mM EDTA + 5g/L BSA</td>
</tr>
<tr>
<td>MACS CD4+ T Cell Isolation Kit II</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-155</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>EasySep&reg; Magnet</td>
<td>equipment</td>
<td>Stem Cell Technologies</td>
<td>18000</td>
<td>		</td>
</tr>
<tr>
<td>5 mL polypropylene tube</td>
<td>equipment</td>
<td>Falcon</td>
<td>352063</td>
<td>		</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Reagent</td>
<td> Invitrogen</td>
<td>22400-089</td>
<td>with 25 mM HEPES</td>
</tr>
<tr>
<td>Pooled human serum</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>drawn from healthy donors, heat inactivated and filtered</td>
</tr>
<tr>
<td>Pen Strep</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>1570-063</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>500mL 0.22 micron bottle top filter</td>
<td>equipment</td>
<td>Nalgene</td>
<td>595-3320</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>48-well plate</td>
<td>equipment</td>
<td>Costar</td>
<td>3548</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>37&deg;C CO2 incubator</td>
<td>equipment</td>
<td>Sanyo Scientific</td>
<td>MCO-18AIC</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>20 mg/mL synthetic peptide</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>Custom synthesis from any peptide vendor</td>
</tr>
<tr>
<td>5 mL FACS tube</td>
<td>equipment</td>
<td>Falcon</td>
<td>352008</td>
<td>Polystyrene</td>
</tr>
<tr>
<td>Peptide loaded class II tetramer (as described)</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>Prepared by the BRI tetramer core</td>
</tr>
<tr>
<td>Anti-CD3</td>
<td>Reagent</td>
<td>BD pharmingen</td>
<td>347344</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Anti-CD4</td>
<td>Reagent</td>
<td>eBioscience</td>
<td>17-0049-73</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Anti-CD25</td>
<td>Reagent</td>
<td>eBioscience</td>
<td>11-0259-73</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Facs Calibur Flow Cytometer</td>
<td>equipment</td>
<td>BD Biosciences</td>
<td>342975</td>
<td>Or equivalent</td>
</tr>		</tbody>
		</table>
		</div>

visualizing antigen specific cd4+ t cells using mhc class ii tetramers

主要组织相容性复合体（MHC）Ⅱ类四聚体可以直接可视化的抗原特异性CD4 + T细胞流式细胞仪。此方法依赖于非常具体的肽载入MHC和相应的T细胞受体之间的互动。虽然单一的MHC /肽分子的亲和力低，交叉连接链霉亲和的MHC /肽复合物增加亲和力的互动，使染色试剂的使用。由于CD4 + T细胞（〜1 30万为一个单一的特异性）的频率相对较低，本试验利用体外扩增步骤，以提高其检测阈值。纯化外周血单个核细胞经Ficoll底图。然后由消极的选择，使用生物素抗体鸡尾酒和抗生物素标记的磁珠分离CD4 +细胞。使用贴壁细胞的CD4细胞的一小部分，作为抗原提呈细胞，CD4 + T细胞在媒体扩大，加入的抗原肽和IL - 2。孵化一小时，随后在37℃染色使用表面抗体，如抗CD4，抗CD3和抗CD25扩大细胞与相应的Ⅱ类四聚体染色。标签后，细胞可以直接通过流式细胞仪分析。扩大CD4 +细胞中的四聚体阳性细胞通常形成一个独特的人口。四聚体阳性细胞通常CD25 +调节性和经常的CD4高。由于背景的四聚体染色水平可以有所不同，应该始终是积极的染色结果相比，相同的细胞与一个不相关的四聚体染色。这一基本检测的多种变化是可能的。四聚体阳性细胞可能为进一步的表型分析，列入酶联免疫斑点或增殖实验，或其他二次检测进行排序。一些团体也展示了合作，使用四聚体的抗细胞因子或抗FOXP3抗体染色。

Watch this Scientific Journal Video about Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers at JoVE.com

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

此过程演示的纯化和体外扩增抗原特异性CD4 +从整体外周血T细胞及其可视化使用MHC II类四聚体。四聚体，允许直接与单个抗原特异性T细胞和定义MHC II类限制性的可视化。

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

visualizing-antigen-specific-cd4-t-cells-using-mhc-class-ii-tetramers

Research

JoVE Journal

Biology

21.6K 次观看.  Benaroya Research Institute. 此过程演示的纯化和体外扩增抗原特异性CD4 +从整体外周血T细胞及其可视化使用MHC II类四聚体。四聚体，允许直接与单个抗原特异性T细胞和定义MHC II类限制性的可视化。

视频: Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

科研

生物学

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

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

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

cd4+ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...