作者衷心感谢 Prof. 和 Dr. 克莱尔 Westhorpe 的工作, 因为他们在开发早期的迭代模型中扮演了关键的角色。作者还要感谢 AMREP 流式细胞仪核心的分单核分子子集和阿尔弗雷德医院传染病组的临床研究护士招募艾滋病毒 + 个人的一些研究。作者衷心感谢伯内特研究所收到的维多利亚运营基础设施支持计划的这项工作的贡献。TAA 得到 RMIT 大学校长博士后奖学金的支持。这项工作得到了澳洲项目补助金1108792授予 AJ 和 AH。传统知识得到 nih 资助 nih K08AI08272、nih/NCATS 补助金 UL1TR000124。

注意: 所有使用人体生物样本的实验都是在墨尔本的阿尔弗雷德医院人类伦理委员会的伦理批准下进行的。所有实验均在第二类生物安全柜中进行, 除非指定。#34;P rewarmed 和 #34; 指在水中加热到37和 #176 的试剂; C. 
 1. i 型纤维状胶原凝胶的制备: 1 天 <ol> <li> 通过依次添加和混合35.7 毫米氢氧化钠、0.71 x M199、4.58 mm 乙酸和1.71 毫克/毫升 i 型纤维胶原制成5毫升聚苯乙烯制备聚合胶原凝胶管按 表 1 . 
	注意: 为了阻止胶原蛋白和 #39;p ockets 和 #39 的形成, 在凝胶混合物中, 确保胶原蛋白混合, 并轻轻移5倍。每测试条件为4-6 凝胶显微镜或15凝胶每测试条件流式细胞仪. </li>
	<li> 一次好的混合, 分50和 #181; 胶原凝胶混合物, 每一个不育的 flat-bottomed 96-良好的组织培养板。不要使用外部行和列, 但用200和 #181 填充这些数据; 1x Dubecco 和 #39 磷酸盐缓冲盐水 (PBS), 以保护凝胶免受干燥。在37和 #176 孵育板; C/5%co 2 用于2小时, 以允许胶原蛋白聚合. 
	注意: 将板材直接放在孵化箱的清洁金属架上, 以确保均匀的热量分配. </li>
	<li> 以下孵育, 用150和 #181 覆盖凝胶; L 1x 补充 M199 (见 材料表 ), 孵育5天直到使用. </li>
</ol> 2。扩展存储的内皮: 1 天 <ol> <li> 标记和涂上10厘米直径的培养皿, 1 毫升50和 #181; 在 1x PBS 中稀释的 g/毫升纤维连接蛋白, 室温下孵育10分钟. </li>
	<li> 解冻等分的人脐静脉内皮细胞 (内皮, 1.0 x 10 6 细胞) 在10毫升 M20. 
	注: 内皮应按先前描述的 21 编写, 并在低通道数 (和 #60; 段落 4) 中使用。内皮可替换为人冠状动脉内皮细胞. </li>
	<li> 重内皮在10毫升 M20, 并添加到纤维连接蛋白涂层碟, 在增加细胞之前, 吸取过多的纤维连接蛋白, 和文化汇合 (约5天), 取代媒体在3天. </li>
</ol> 3。胶原凝胶培养内皮单层: 5 天 <ol> <li> 在5天, 从培养皿中吸取培养基分离内皮, 然后用10毫升无血清 M199 洗去含血清的培养基. </li>
	<li> 在 M199 中添加5毫升0.05% 胰蛋白酶/0.53 EDTA, 内皮, 室温下孵育1-2 分钟, 轻轻晃动, 直到细胞分离. </li>
	<li> 一旦分离, 用5毫升 M20 快速冲洗培养皿, 并将含有细胞的培养基传送到10毫升管. </li>
	<li> 在室温下离心取样 300 x g, 吸气上清, 重细胞在200和 #181; L M20, 用例计数细胞. </li>
	<li> 将单元格重为 2.0 x 10 5 M20 中的单元格/mL 从96井板 (步骤 1.3) 中吸取凝胶上的 M199, 并添加100和 #181; 悬浮内皮 (2.0 x 10 4 细胞) 对上述每种胶原凝胶 (步骤 1.2)。文化板材在3天在37和 #176; C/5%co 2 . 
	注: 内皮单层的完整性可在3天后经硝酸银染色后确认为 22 或在本阶段对紧密接合蛋白 23 进行免疫组化。必须为此目的准备额外的凝胶. </li>
</ol> 4。内皮单分子膜的活化和分离/细胞活化: 8 天 <ol> <li> 在天 8, 激活每个内皮单层之前, 通过吸取 M20 介质覆盖凝胶和添加100和 #181; L 的 10 ng/毫升人 TNF 和 #945; 在 M20 每凝胶。孵育在37和 #176; C/5%co 2 为 4 h. 
	注意: 如果需要, 非激活的内皮条件也可能包含在控件中. </li>
	<li> 在 4 h 内皮激活步骤中, 使用磁珠技术将单核细胞从 PBMCs 中分离出来, 按照制造商和 #39 的指示对电池进行负选择。在此步骤中, 至少应使用 6.5 x 10 6 PBMCs, 以便可靠地恢复一个条件所需的 3.0 x 10 5 单核细胞, 因为单核细胞通常占约10-15% 的 PBMCs. 
	注意: 为了评估单核细胞在单核轮回和泡沫形成前的作用, 在这个阶段可以激活单核。单核细胞可以从解冻 PBMCs, 如果需要 ( 即 , 储存的病人样本) 隔离。可为除凝胶 ( 图 1 ) 而准备的单核细胞群分类分离。
</ol> 5。初级人类单核细胞的轮回: 8 天 <ol> <li> 测量单核 M199, 清除 TNF 和 #945;-含培养基和洗涤凝胶两次与100和 #181; 通过添加和移除媒体, 我对此表示满意....。在洗涤之后, 增加 2.0 x 10 5 新鲜地被隔绝的或解冻和洗涤的被保存的 PBMCs 或 5.0 x 10 4 新孤立单核细胞或纯化单核细胞亚群到内皮单分子膜在100和 #181; M20孵育1小时, 在37和 #176; C 和 5% CO 2 , 允许单核细胞向凝胶中的前向轮回。最好是让6口井为每一个实验条件检查. 
	注: 为了评估自体血清对轮回和泡沫细胞形成的影响, 孵育细胞的 M20 含有所需的热灭活供血的浓度, 并与人类血清控制的条件进行比较。在这个阶段, 除了单核细胞之外, 还可以添加白血球。如果调查特定脂质/脂类 ( 如 、高密度脂蛋白、oxHDL) 的影响, 请执行所有以下步骤, 其中含有20-50 和 #181 的无血清培养基; 克/毫升脂. </li>
	<li> 经过1小时的前向轮回, 通过洗涤凝胶两次收集 non-transmigrated 细胞与100和 #181; l prewarmed 1 毫米 EGTA 在 1x PBS, 和一次与100和 #181; M199 (相同的离心条件), 汇集清从每个井实验条件 (通常6口井) 变成1.5 毫升离心管在冰上。离心 non-transmigrated 细胞在4和 #176; C, 300 x g 为 5 min 和重在30和 #181; L 1x PBS. </li>
	<li> 计数上清中收集的单元格以确定正向轮回单元格的数量. </li>
	<li> 向前轮回单元格的百分比按以下方式确定: 
	<img alt="Equation 1" src="/files/ftp_upload/56293/56293eq1.jpg"/> <img alt="Equation 2" src="/files/ftp_upload/56293/56293eq2.jpg"/> <img alt="Equation 3" src="/files/ftp_upload/56293/56293eq3.jpg"/> </li> <li> 覆盖已清洗含有轮回细胞的凝胶100和 #181; L M20 和孵育 48 h. </li>
	<li> 48 小时后, 收集上清液和冲洗 non-transmigrated 细胞两次, 100 和 #181; L prewarmed 1 毫米 EGTA/PBS, 收集和汇集的上清从每个条件的步骤 5.2. 
	注: 反转轮回细胞的表型可在这些细胞上测试ollowing 标准流式细胞仪染色协议. </li>
	<li> 4 和 #176 的离心细胞; 30 和 #181 中的5分钟和重细胞 300 x g; 1x PBS计数细胞, 通过台盼蓝染色确定生存能力. </li>
	<li> 使用下列公式确定反向轮回单元格的百分比: 
	<img alt="Equation 4" src="/files/ftp_upload/56293/56293eq4.jpg"/> 注意: 对正向轮回单元的总数量进行核算, 可以更准确地显示反向轮回, 因为它不太可能向前轮回100%. </li>
	<li> 通过显微镜进行分析, 通过增加100和 #181 来固定凝胶, 并将2% 甲醛 (最终浓度) 对每一个井, 盖在铝箔和存储在4和 #176; C 直到分析。对于流式细胞术, 不要在这个阶段修复细胞。见下面的协议显微镜 (6 节) 或流式细胞仪 (第7节) 分析. 
	注意: 甲醛有毒, 腐蚀性强;使用个人防护设备 (PPE)。在添加甲醛时应注意, 但是, 由于使用的浓度较低, 此步骤不需要在油烟罩中执行. </li>
</ol> 6。泡沫细胞和巨噬细胞的显微定量 (油红色 O 染色) <ol> <li> 取出甲醛, 用100和 #181 清洗凝胶; 50% (v/v) 甲醇为5分钟, 然后100和 #181; l 78% (v/v) 甲醇为 15 min. 
	注: 染色步骤可在实验室工作台上进行, 而不是在第二类生物危害柜中进行. </li>
	<li> 添加100和 #181 的脂液滴染色; 新准备的 0.2% (w/v) 油红色 O (参见 材料表 详细信息), 在室温下为1小时. </li>
	<li> 用100和 #181 洗涤凝胶4次, 去除多余的污渍; 78% (v/v) 甲醇, 每次洗涤后, 抽吸和丢弃上清液. </li>
	<li> Counterstain 在室温下为15分钟, 100 和 #181; L 姬姆萨, 稀释1:10 的蒸馏水. </li>
	<li> 吸姬姆萨渍, 用清水冲洗一次凝胶. </li>
	<li> 将凝胶从盘中分离出来, 用21克针将油井边缘化, 斜面朝向外, 围绕凝胶边缘. </li>
	<li> 要在显微镜滑轨上安装凝胶, 请在 2.54 cm x 1.5 厘米的双面胶带上打两个孔 (直径 6.35 mm)。将胶带固定在标准显微镜一侧, 从顶部取下防护涂层. 
	<ol> <li> 使用传输吸管向磁带中的孔添加一滴水。使用镊子, 轻轻地转移凝胶到磁带上的孔和盖1.5 大小的玻璃片。轻轻地按片, 将其粘附到磁带上. </li>
	</ol> </li> <li> 在倒置显微镜下使用40X 目标进行差分干涉对比亮场显微镜检查. 
	<ol> <li> 使内皮单层在40X 放大和滚动 #39; 进入和 #39; 凝胶, 计数巨噬细胞/泡沫细胞在整个凝胶的深度。重复三不同的视野位于类似的距离, 从凝胶边缘, 以尽量减少可能的边缘效果。这将确保凝胶深度在计数区域之间保持一致. 
		注: 将凝胶中的细胞作为泡沫细胞 (被定义为含有和 #62 的细胞; 1/3 的细胞质为油红色 O 染色的脂滴) 或2小时内在幻灯片上的安装 ( 图 2 )。泡沫细胞的形成被表达为相对于迁移细胞总数的泡沫细胞的百分比, 并表示为每条件6凝胶的3个视场中的中位数, 因此每个条件的18测量中位数。典型实验中的原始单元格计数的示例如 表 2 所示. 
		注: 显微镜下对细胞作为泡沫细胞与巨噬细胞的分类是主观的, 因此可以依赖于研究者。在临床研究中, 实验是在较长时间内进行的, 对所有计数都是很重要的。在 图 2 中提供了凝胶中的泡沫细胞和噬细胞的例子. </li>
	</ol> </li> </ol> 7。流式细胞术对轮回细胞的分析 <ol> <li> 型 transendothelial 迁移后的泡沫细胞和巨噬细胞的特征, 通过增加100和 #181 来消化凝胶; L 37 和 #176; C prewarmed 1 毫克/毫升胶原酶 D 稀释在 M199每个井和孵化20分钟, 在37和 #176; C/5%co 2 . 
	注: 将96井板直接放在孵化箱的清洁金属架上, 以确保均匀的热量分配. </li>
	<li> 以下孵育, 使用200和 #181 浸渍凝胶; L 吸管尖和孵育20分钟, 在37和 #176; C/5%co 2 . </li>
	<li> 一旦完全消化, 过滤和池的消化复制凝胶通过35和 #181; m 尼龙网格盖在冰上, 并清洗一次与外地资产管制署洗涤 (见 材料表 ) 在4和 #176; C, 300 x g. 重100和 #181 的细胞;洗涤. </li>
	<li> 使用标准流式细胞仪协议, 通过特定于 CD45 的荧光共轭抗体、活/死细胞标记和表面/细胞内表型或功能性标记来孵育产生的细胞. 
	注: 使用适当的荧光和 Ig 补偿珠准备补偿用管。提取的细胞也可以收集到玻片的免疫荧光显微镜 cytospinning。另外, 我们已经成功地收集了使用该协议的细胞, 通过成像流式细胞仪进行评估. </li>
	<li> 使用流式细胞仪获取单元格, 并根据需要执行补偿. 
	注: 因为泡沫细胞/巨噬细胞可能不会形成单个群体使用光散射特性, 并可能在大小和形状上有很大差异, 设置自由向前散射 (FSC) 和侧散射 (SSC) 门, 以捕获所有迁移的细胞, 如图所示强类 = "xfig" > 图 3A . </li>
	<li> 在获得后, 使用流式细胞仪软件进行数据分析。若要标识迁移的单元格, 请首先选择 "活/死" 标记的单元格为负数, 如 图 3B 所示。接下来, 通过在 ssc 区域与 ssc 高度图上显示的单元格周围创建一个紧密的栅极来执行单细胞判别. </li>
	<li> 选择 CD45 + 单元格和门在 FSC/SSC 图中存在的迁移细胞的主要种群, 因为这些是巨噬细胞/泡沫电池. </li>
</ol>

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD14++CD16++monocytes+and+cardiovascular+outcome+in+patients+with+chronic+kidney+disease.">CD14++CD16+ monocytes and cardiovascular outcome in patients with chronic kidney disease.</a> Eur. Heart J. 32 (1), 84-92 (2011).</li></ol>

作者没有什么可透露的。

这里描述的协议提供了一个多才多艺的和生理学相关的方法来评估的 atherogenicity 单核细胞从人的临床组, 结合单核的轮回和泡沫形成。这种模式提供了比其他泡沫细胞形成的方法的优势, 因为它考虑到单核细胞的轮回对泡沫形成的影响, 并允许测量反向轮回6除了固有的在外源性因素存在或缺乏的情况下, 单核细胞倾向于成熟成泡沫。此外, 内皮活化的作用也被考虑到, 因为我们已经表明, 内皮细胞活化上调氧化的脂类与泡沫电池形成14。最后, 通过显微镜和流式细胞仪可以量化和表征单核的表型 (与斑块回归相关的特性)。
由于三维性质的轮回到凝胶, 识别和评分轮回细胞作为泡沫细胞或巨噬细胞可能是困难的单元格刺字到不同的水平在凝胶19。为了帮助识别泡沫细胞, 必须始终使用新鲜油红色 O 染色。另外还要注意的是, 不同的体内疾病状态或体外条件可能会改变单核细胞在凝胶中的轮回。我们使用活细胞成像技术来确定从 HIV 感染的个体中的单核刺字到凝胶中的浅点, 并以不同于未感染个体的速度19。此外, 泡沫细胞的形成与较少的单核细胞运动在凝胶中, 所以在一个特定的 z 段, 单元格倾向于移动在一个相对于非 HIV 感染的人的细胞, 倾向于迁移在一条比较直线的方向底部的凝胶。这些发现提高了类似的现象的可能性发生在实验中使用人类病人样本从其他疾病状态与增加单核 atherogenicity。
当执行流 cytometry-based 分析时, 在使用活/死细胞标记 (图 4B) 评估生存率时, 通常要观察到恢复的细胞数量中的相当大比例的死细胞。这些细胞主要是 CD45 的-内皮, 在胶原蛋白基质中提取单核细胞的过程中被破坏。这个过程是特别苛刻的内皮单层, 但不损害分的轮回细胞。
这种模型不同于其他的, 因为在培养基中不需要人工氧化的外源脂质来驱动单核细胞衍生泡沫的形成。相反, 这种检测中的泡沫细胞的形成受培养基中的血清的影响, 从而可以对临床样品中可溶性因子的离散效应进行评估。因此, 我们已经表明, 从感染 hiv 病毒的年轻的19或老年 hiv 感染者的血清中控制单核细胞的培养可以促进泡沫形成, 表明可溶性因子能独立促进泡沫细胞形成。由于测定结果受可溶性成分的影响, 必须用一组单独的人血清进行实验, 目的是确定不同个体的单核细胞动脉粥样硬化特性的内在差异, 以规范控制条件。这种测定方法也可用于评价临床样本中特定脂类的作用 (图 3)。例如, 我们发现, 含有孤立脂类的介质的单核细胞的培养, 如来自已知脂质功能障碍的个体的高密度脂蛋白,24也促进了泡沫形成。在这种情况下, 单核细胞从一个单一的健康个人或群体可以使用的存在血清或血清因素的测试对象派生。因此, 这个模型允许特定的问题被问及关于溶解和细胞成分对泡沫细胞形成的离散效应。
该方法利用内皮作为冠状动脉内皮的模型, 因为在获得大量的低通道数原发性冠状动脉细胞方面存在实际困难。然而, 我们比较了使用人冠状动脉内皮细胞或内皮的化验结果, 观察到单核细胞的轮回或泡沫形成的小差异 (数据没有显示), 表明在这个系统中使用内皮是一个可接受的替代品。手动计数泡沫细胞是一个限制因素, 在这个模型, 因为它可能会引入操作员的偏见。因此 , 所有安装在幻灯片上的样品都必须作者蒙蔽 , 以消除偏差的可能性。然而, 我们已经通过人工计数和成像流式细胞仪对泡沫细胞形成进行了比较, 发现这些方法具有相似的结果19。该模型的一个关键限制是单核细胞黏附和轮回发生在没有与生理血流相关的剪切力体内。我们推测, 剪切流将主要影响单核细胞的轮回, 而不是在矩阵内的后续泡沫单元形成;然而, 这必须考虑时, 解释的结果, 从这个化验。这一分析也没有模型的影响, 平滑肌细胞的单核来源泡沫细胞形成, 这是一个生理限制系统;然而, 这在所有条件下都是一致的, 允许将单核细胞的离散动脉粥样硬化电位与不同疾病状态进行比较。
总之, 该模型提供了一个多用途和生理学相关的方法, 量化单核细胞的轮回和泡沫形成的人样本在不同的疾病状态前体内。该模型进一步应用于评估单核细胞的倾向, 形成泡沫电池的条件下, atherogenicity 与增加风险的 CAD, 如肥胖25, 糖尿病26和慢性肾脏疾病27。因此, 这种模型也可以优化, 以用于疾病的状态, 而不是动脉粥样硬化, 细胞轮回可能影响疾病的发病机制。

单核细胞轮回是动脉粥样硬化斑块发展的关键一步, 可能导致血栓、中风和心肌梗塞。动脉粥样硬化斑块发育的脂肪条纹, 一般目前在低振荡血流在中到大的动脉, 在那里沉积脂质有助于内皮细胞活化和局部炎症1。单核细胞通过单核趋蛋白 (如 CCL2) 和刺字进入内膜2, 被吸收到脂肪条纹中的内皮。在轮回之后, 单核细胞可能形成动脉粥样硬化的、脂质的巨噬细胞, 称为脂质的摄取、脂质合成、胆固醇外流调节或上述因素的综合作用的结果。单核细胞也可以在循环中积聚脂质, 并有一个泡沫状的表型, 可能是发泡细胞形成的诱因3,4。泡沫细胞是脂肪条纹和早期动脉粥样硬化斑块的决定性特征, 其形成受脂质和炎症介质的影响5。另外, 单核细胞有能力逆转刺字从动脉进入血流6, 从而消除脂质从内膜和行动, 以保持健康的动脉。
确定单核细胞在动脉内皮刺字的倾向, 在内膜内形成泡沫细胞, 或逆转刺字并携带脂质, 是了解细胞活化在增加动脉粥样硬化风险。小鼠模型的 cad, 如动脉粥样硬化是重要的阐明 real-time在体内信息的脂肪条纹/动脉粥样硬化斑块的发展。然而, 这些模型需要基因改变这些动物的自然胆固醇处理能力通常加上剧烈的改变饮食 (如载脂蛋白/西方型饮食模式)7,8, 从而,诱导 non-physiological 积累的循环脂水平, 推动斑块的发展。这些模型可能与慢性炎症性人类疾病的相关性有限, 如 HIV 感染, 而不与增加循环胆固醇或低密度脂蛋白 (LDL) 水平有关。此外, 人与小鼠之间单核细胞生物学的差异, 使得对单核细胞亚群 (如中间单核细胞 (CD14++CD16+)) 的相关性的免疫学问题的测试,9困难.这是重要的, 当研究的机制驱动心血管疾病作为中间单核细胞计数独立预测心血管事件10,11。虽然化验存在顺序测量单核细胞的轮回或泡沫形成的隔离, 没有体外试验已验证, 以量化两方面的早期动脉粥样硬化使用相同的细胞从临床组。Transwell 模型利用一个改进的博伊登两室系统, 其中细胞被加载到顶部室和刺字跨多孔塑料屏障或细胞单层到一个较低的会议厅, 通常包含具有趋化因子12的媒体,13. 虽然广泛用于分析白细胞的轮回, 但这些模型一般不包含一层代表内膜, 导致轮回细胞迁移到溶液中, 不允许测量泡沫细胞相同细胞的形成或反向轮回。相反, 泡沫细胞形成的模型不解释任何 transmigratory 引起的单核细胞的改变或内皮激活的影响, 这是已知的贡献泡沫电池形成14。此外, 这些系统诱导泡沫细胞形成从附着在细胞培养板上的饱和浓度的外源氧化低密度脂蛋白 (oxLDL)15,16, 一个关键的诱导剂泡沫细胞的形成。这些模型中使用的 LDL 通常被 non-physiologically 相关的过程 (如丘索4治疗17) 所氧化, 因此, 使用这些模型来质疑研究的生理学重要性。
在这里, 我们描述了一个定量分析, 量化单核细胞的轮回和泡沫细胞的形成, 不需要添加外源性 oxLDL, 从而更好地模拟单核在泡沫细胞形成中的作用。这个模型最初是由威廉. 穆勒教授 (西北大学, 芝加哥)18开发的, 并且在我们的实验室中得到了进一步的改进, 以评估ex 体内atherogenicity 下单核细胞在 non-activating 下的分离与患动脉粥样硬化的风险增加相关的疾病, 如 HIV 感染19以及衰老的20的患者的条件。该模型还提供了一个平台, 回答基本的生物学问题的倾向, 不同的单核细胞亚群形成泡沫电池20, 细胞因子, 如 TNF 对泡沫细胞形成的影响, 内皮激活14, 以及在凝胶中轮回的深度和速度等单核细胞的迁移特性19。此外, 单核细胞的轮回和泡沫形成可以量化使用标准显微镜, 活细胞成像, 流式细胞仪和成像流式细胞仪, 因此, 提供了一个多用途的方法来评价单核在动脉粥样硬化的作用。

<table border="0" cellpadding="0" cellspacing="0" width="1187">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Gel preparation reagents</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>221465-500G</td>
			<td>0.1 M NaOH diluted in H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">10X M199</td>
			<td>Sigma-Aldrich</td>
			<td style="width:111px;">M0650</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">AcCOOH</td>
			<td>Sigma-Aldrich</td>
			<td>695092-100ML</td>
			<td>20 mM Acetic acid diluted in H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cultrex Bovine Collagen I</td>
			<td>R&#38;D Systems</td>
			<td>3442-050-01</td>
			<td>Type I Fibrous Collagen</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">M199</td>
			<td>Life Technologies</td>
			<td>11150-059</td>
			<td style="width:595px;">M199 media containing Earle&#39;s salts, L-glutamine and 2.2 g/L Sodium Bicarbonate. 
			Media supplemented with 100 &#181;g/mL L-glutamine&#160; and 100 U/mL penicillin/streptomycin&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">M20</td>
			<td></td>
			<td></td>
			<td style="width:595px;">Supplemented M199 containing 20% heat-inactivated pooled or donor serum. 
			Individual lipid species such as LDL can be added to M199.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">HUVEC</td>
			<td></td>
			<td></td>
			<td style="width:595px;">Primary human umbilical cord endothelial cells (HUVEC) can be isolated from umbilical cords donated with informed consent and ethics approval. Isolated HUVEC may also be purchased commercially.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human coronary artery endothelial cells</td>
			<td></td>
			<td></td>
			<td style="width:595px;">Primary human coronary artery endothelial cells can be isolated from arteries donated with informed consent and ethics approval. Isolated cells may also be purchased commercially.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>BDH Merck</td>
			<td>10093.5V</td>
			<td>Ethylenediaminetetraacetic acid (EDTA) - 0.5 M, pH 8.0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-500G</td>
			<td>Ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA) - 1 mM, pH 8.0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.05% trypsin EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>0.05% trypsin/0.53 EDTA (1X)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>L-glutamine (200 mM)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Penicillin/streptomycin (10,000 units/mL Penicillin and 10,000 &#181;g/mL Streptomycin)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">New born calf serum</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:111px;">16010-142</td>
			<td style="width:595px;">New born calf serum: New Zealand origin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F1056-1MG</td>
			<td>Fibronectin - 50 &#181;g/mL aliquots prepared and stored</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">PBS (1X)</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (10X): Dilute to 1X with sterile H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.1% TNF</td>
			<td>Gibco</td>
			<td>PHC3015</td>
			<td>Recombinant human TNF - Reconstituted in H20 and stored in 10 &#181;g/mL aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low-density lipoprotein</td>
			<td>Merck Millipore</td>
			<td>LP2-2MG</td>
			<td>Low-density lipoprotein (LDL)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Microscopy</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1 or 2% formaldehyde</td>
			<td>Polysciences</td>
			<td>4018</td>
			<td>1 or 2% formaldehyde diluted with sterile H20</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50% and 78% methanol</td>
			<td>Ajax Finechem</td>
			<td>318-2.5L GL</td>
			<td>50% or 78% v/v methanol, diluted with H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil Red O stain</td>
			<td>Sigma-Aldrich</td>
			<td>O0625-25G</td>
			<td>Dilute to 2 mg/mL in 22% 1M NaOH and 78% (v/v) methanol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope slides</td>
			<td>Mikro-Glass</td>
			<td>S41104AMK</td>
			<td style="width:595px;">Twin frosted 45 degree ground edge microscope slides (25 X 76 mm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover slips</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>MENCS224015GP</td>
			<td>22 x 40 mm #1.5 size glass cover slips</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double-sided tape</td>
			<td>3M Scotch</td>
			<td>4011</td>
			<td>Super strength exterior mounting tape (25.4 mm x 1.51 m)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Giemsa stain</td>
			<td>Merck Millipore</td>
			<td>1.09204.0500</td>
			<td>Giemsa&#39;s azur eosin methylene blue solution (dilute stock 1:10 in H20)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Hole punch</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;">Hand-held single hole punch (6.35 mm punch)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Flow cytometry</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase D</td>
			<td>Roche Diagnostics</td>
			<td>11088858001</td>
			<td>Collagenase D diluted in M199 media to 1 mg/mL&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 &#181;m nylon mesh capped polystyrene FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>35 &#181;m nylon mesh capped polystyrene FACS tubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Fixable Yellow Dead Cell stain</td>
			<td>Life Technologies</td>
			<td>L34959</td>
			<td>Live/Dead Fixable Yellow Dead Cell stain</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">FACS wash</td>
			<td></td>
			<td></td>
			<td>Prepare by mixing 1 X PBS-, 2 mM EDTA and 1% New born calf serum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Plasticware</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate</td>
			<td>Nunclon</td>
			<td>167008</td>
			<td>Delta surface flat-bottomed 96 well plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm Petri Dish</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Sterile 10 cm Petri dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td>Eppendorf tubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco Scientific</td>
			<td>222-20S</td>
			<td>Sterile transfer pipette (1 mL, large bulb)</td>
		</tr>
	</tbody>
</table>

quantification of monocyte transmigration and foam cell formation from individuals with chronic inflammatory conditions

冠状动脉疾病 (CAD) 是全球发病率和死亡率的主要原因。动脉粥样硬化是 CAD 的一个主要原因, 它是由先天免疫单核细胞的轮回所引发的, 它被称为脂肪条纹的脂质的炎症部位, 它们存在于中到大动脉的动脉壁上。动脉粥样硬化早期病变的主要致病特征是单核细胞的成熟, 它会迁移到动脉形成泡沫或脂质巨噬细胞。大量的证据支持这样的假说: 动脉粥样硬化的风险是由慢性炎症性疾病, 如类风湿关节炎和 HIV, 以及一般的老化, 以及这种风险是由单核细胞预测激活.尽管鼠标模型提供了一个很好的平台来研究单核细胞在动脉粥样硬化体内的作用, 但它们需要对天然胆固醇代谢进行基因改变和对正常小鼠饮食的剧烈改变, 并且对人类共疾病的动脉粥样硬化影响研究。这促使我们开发了一个人的体外模型, 以测量孤立于定义疾病状态的个体的单核细胞的动脉粥样硬化潜能。目前, 人类的体外模型是限制的, 他们评估单核细胞的轮回和泡沫形成的孤立。在这里, 我们描述了一个协议, 其中单核细胞从病人的血液刺字跨人的内皮细胞膜到1型胶原基质, 他们的倾向成熟成泡沫细胞存在或没有外源性脂质测量。该议定书已得到验证, 用于使用从艾滋病毒感染者和老年艾滋病毒感染者身上纯化的人单核细胞。这个模型是多才多艺的, 并且允许单核细胞的移居和泡沫形成被评估使用显微镜或流式细胞仪并且允许评估动脉粥样硬化因素存在于血清或血浆。

量化单核细胞轮回
单核细胞被添加到模型中, 如图 1所述, 每个条件都准备了六凝胶。单核细胞为6凝胶每个施主 (即, 5.0 x 104单核细胞每凝胶6凝胶 = 3.0 x 105单核细胞每个施主) 是悬浮到最后的容量600µL 的 M199 媒体含有所需的血清/分离脂质。细胞 (i. e, 100 µL 每凝胶 = 5.0 x 104单核) 然后 aliquoted 到凝胶和孵育为 1 h, 以方便单核轮回。在轮回之后, non-transmigrated 细胞被计算为以上。在此示例中, 恢复了 1.5 x 104 non-transmigrated 单元格。使用步骤5.4 中描述的公式确定了向前轮回的百分比:
<img alt="Equation 5" src="/files/ftp_upload/56293/56293eq5.jpg"> 
<img alt="Equation 6" src="/files/ftp_upload/56293/56293eq6.jpg">
在 48 h 潜伏期后, 3.6 x 104反向轮回细胞恢复为上述。使用步骤5.8 中的公式确定了反向轮回的百分比:
<img alt="Equation 7" src="/files/ftp_upload/56293/56293eq7.jpg">
正向和反向轮回可以比较的条件使用适当的统计测试和单核细胞制备从多个独立的捐助者, 以确定是否单核细胞的轮回能力改变之间的实验条件.
泡沫细胞形成的评价
明显微镜
氧化脂蛋白是众所周知的促进单核细胞衍生泡沫电池形成在体外相比, unoxidized 血脂15。在这里, 我们证实, oxLDL 的治疗, 通常用于诱导泡沫细胞形成的常规泡沫细胞诱导模型, 提高单核细胞衍生泡沫形成在这个模型的 transendothelial 迁移和泡沫细胞形成与本地 LDL 相比。随单核细胞的轮回和泡沫细胞的形成在凝胶孵育与 M199 含有50µg/毫升本机或丘索4-氧化 LDL (见步骤 5.1,图 1), 通过显微镜进行分析。在代表性实验中观察到的泡沫细胞和巨细胞的例子如图 2所示。细胞被计数在三不同的领域每胶凝体, 通过聚焦在内皮单层, 并且逐渐聚焦入更加深刻入胶凝体。每个捐赠者的细胞计数记录在表 2中, 用于对单个捐赠者进行对比, 以比较在氧化和本地 LDL 反应中形成的泡沫细胞的百分比。从 n = 12 独立计数的聚合数据显示在图 4中, 以确认此模型中 oxLDL 的单核细胞的孵化会导致更多的泡沫形成, 而不是仅靠本地低密度脂蛋白或 M199 介质来孵化的情况。
流式细胞仪
为了确定移植细胞的表型, 从消化凝胶中提取细胞, 用标准流式细胞仪进行标记。细胞被标记为活/死细胞标记和抗体特异的 CD45 和其他表面表型标记使用标准流式细胞仪协议。补偿控制也必须按照标准流式细胞术技术进行充分补偿。迁移的单元格是封闭的, 如图 3中所示, 代表10独立的实验。使用活细胞/死细胞生存能力检测 (图 3B) 和双峰排除在单细胞分析 (图 3C) 中。然后, 将单元格作为 CD45+进行封闭, 以排除内皮 (因为这些单元格是 CD45-,图 3D)。然后通过前向散射/侧散射判别 (图 3E) 对主要种群进行门控, 并将感兴趣的受体的平均荧光强度 (FMO) 与荧光减去一 (或同种) 控制相比较来确定 (图 3F)确定阳性细胞的表达 (ΔMFI) 或百分比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56293/56293fig1.jpg"> 
图 1:体外单核细胞轮回和泡沫形成的模型.( A) PBMC, (B) 总单核细胞或 (C). 在96井板中添加的 i 型纤维状胶原蛋白凝胶, 并覆盖一层活化的初级人类脐静脉内皮细胞 (内皮) , 其完整性可以评估银染色(D) 和允许 (E) 刺字1小时存在的血清或含脂介质。非轮回细胞被计算为 ( F), 并且凝胶被孵化为另外48小时, 具有相同的血清/脂类介质。下面的孵化, (G) 反向迁移的细胞计数和泡沫细胞的百分比在凝胶是由 ( H) 相衬显微镜后染色, 油红色 O 或迁移细胞的表型由 ( I) 流式细胞仪确定。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56293/56293fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56293/56293fig2.jpg"> 
图 2: 泡沫电池和巨噬细胞的例子体外单核细胞轮回和泡沫形成的模型.Representat细胞的例子被计分作为泡沫细胞 (白色箭头) 和噬细胞 (黑箭头) 在姬姆萨染色以后形象化细胞和油红色 O 染色形象化脂滴由被提取的凝胶的相对比显微镜使用倒置显微镜在40X 放大倍数。缩放栏 = 20 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56293/56293fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56293/56293fig4.jpg">
图 3: 流式细胞术轮回细胞的门控策略轮回细胞是型的特征, 流式细胞术后提取细胞从凝胶。单元格由 (A) (FSC/SSC) 封闭 (B) 活细胞, (C) 单细胞, (D) CD45+, (E) FSC/SSC 和 (F) 受体表达与同种或荧光减去一 (FMO) 控制。兴趣受体的表达被定义为平均荧光强度 (同种) 和表达的水平。ΔMFI = 污点 (多边基金会) 减去同种/FMO 控制。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56293/56293fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56293/56293fig3.jpg">
图 4: 外部添加 oxLDL 和低密度脂蛋白对单核细胞衍生泡沫形成的影响.泡沫细胞的形成是确定的条件下, 单核从单一的捐助者孵化培养基 (M199) 或50µg/毫升 unoxidized 低密度脂蛋白 (ldl) 或铜 (II) 硫酸盐氧化 ldl (oxLDL) 添加到凝胶 post-transmigration。显示了12不同站点的计数。泡沫细胞以总迁移细胞的百分比表示。在条形图中显示中值和分范围, 并使用非参数曼-惠特尼 U 测试进行比较。p & #60; 0.001, *** 和 #60; 0.0001。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56293/56293fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>试剂</td>
			<td>股票]</td>
			<td>最后]</td>
			<td>60凝胶容积 (µL)</td>
		</tr>
		<tr>
			<td>naoh</td>
			<td>100毫米</td>
			<td>35.7 毫米</td>
			<td>1071</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>10x</td>
			<td>0.71x</td>
			<td>213</td>
		</tr>
		<tr>
			<td>AcCOOH</td>
			<td>20毫米</td>
			<td>4.58 毫米</td>
			<td>687</td>
		</tr>
		<tr>
			<td>胶原</td>
			<td>5毫克/毫升</td>
			<td>1.71 毫克/毫升</td>
			<td>1029</td>
		</tr>
		<tr>
			<td>总容积</td>
			<td>-</td>
			<td>-</td>
			<td>3000</td>
		</tr>
	</tbody>
</table>
表 1: i 型纤维胶原凝胶的制备
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="3">ldl</td>
			<td></td>
			<td colspan="3">oxldl</td>
		</tr>
		<tr>
			<td>胶</td>
			<td>数</td>
			<td>泡沫电池1</td>
			<td>迁移的单元格1</td>
			<td>泡沫细胞 (%)2</td>
			<td></td>
			<td>泡沫电池1</td>
			<td>迁移的单元格1</td>
			<td>泡沫细胞 (%)2</td>
		</tr>
		<tr>
			<td>1</td>
			<td>1</td>
			<td>5</td>
			<td>44</td>
			<td>11。4</td>
			<td></td>
			<td>26</td>
			<td>55</td>
			<td>47。3</td>
		</tr>
		<tr>
			<td></td>
			<td>2</td>
			<td>5</td>
			<td>27</td>
			<td>18。5</td>
			<td></td>
			<td>10</td>
			<td>23</td>
			<td>43。5</td>
		</tr>
		<tr>
			<td></td>
			<td>3</td>
			<td>11</td>
			<td>52</td>
			<td>21。2</td>
			<td></td>
			<td>19</td>
			<td>39</td>
			<td>48。7</td>
		</tr>
		<tr>
			<td>2</td>
			<td>1</td>
			<td>5</td>
			<td>34</td>
			<td>14。7</td>
			<td></td>
			<td>9</td>
			<td>31</td>
			<td>29</td>
		</tr>
		<tr>
			<td></td>
			<td>2</td>
			<td>14</td>
			<td>50</td>
			<td>28</td>
			<td></td>
			<td>32</td>
			<td>55</td>
			<td>58。2</td>
		</tr>
		<tr>
			<td></td>
			<td>3</td>
			<td>5</td>
			<td>37</td>
			<td>13。5</td>
			<td></td>
			<td>19</td>
			<td>37</td>
			<td>51。4</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1</td>
			<td>10</td>
			<td>37</td>
			<td>27</td>
			<td></td>
			<td>28</td>
			<td>52</td>
			<td>53。8</td>
		</tr>
		<tr>
			<td></td>
			<td>2</td>
			<td>7</td>
			<td>33</td>
			<td>21。2</td>
			<td></td>
			<td>11</td>
			<td>31</td>
			<td>35。5</td>
		</tr>
		<tr>
			<td></td>
			<td>3</td>
			<td>7</td>
			<td>38</td>
			<td>18。4</td>
			<td></td>
			<td>28</td>
			<td>53</td>
			<td>52。8</td>
		</tr>
		<tr>
			<td>4</td>
			<td>1</td>
			<td>9</td>
			<td>44</td>
			<td>20。5</td>
			<td></td>
			<td>14</td>
			<td>33</td>
			<td>42。4</td>
		</tr>
		<tr>
			<td></td>
			<td>2</td>
			<td>7</td>
			<td>33</td>
			<td>21。2</td>
			<td></td>
			<td>22</td>
			<td>47</td>
			<td>46。8</td>
		</tr>
		<tr>
			<td></td>
			<td>3</td>
			<td>11</td>
			<td>50</td>
			<td>22</td>
			<td></td>
			<td>11</td>
			<td>30</td>
			<td>36。7</td>
		</tr>
		<tr>
			<td colspan="3">中位泡沫细胞 (%)</td>
			<td></td>
			<td>20。8</td>
			<td></td>
			<td></td>
			<td></td>
			<td>47</td>
		</tr>
		<tr>
			<td colspan="3">平均泡沫细胞 (%)</td>
			<td></td>
			<td>19。8</td>
			<td></td>
			<td></td>
			<td></td>
			<td>45。5</td>
		</tr>
	</tbody>
</table>
表 2: 从单核细胞衍生的泡沫细胞与 oxLDL 治疗后的比较, 原电池计数.1每个视场的细胞计数在凝胶。2泡沫细胞百分比 = 泡沫细胞计数/总迁移细胞计数 x 100。*数据是一个实验的代表性, 使用单核细胞从单一的捐助者

Watch this Scientific Journal Video about Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions at JoVE.com

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

我们描述了一项协议, 以测量单核细胞在人的内皮单分子膜的轮回, 并随后成熟成泡沫电池。这提供了一种通用的方法来评估的动脉粥样硬化性质的人单核细胞分离的不同疾病的情况下, 并评估血液中的因素, 可能会提高这种倾向。

慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

quantification-monocyte-transmigration-foam-cell-formation-from

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.2K 次观看.  Burnet Institute. 我们描述了一项协议, 以测量单核细胞在人的内皮单分子膜的轮回, 并随后成熟成泡沫电池。这提供了一种通用的方法来评估的动脉粥样硬化性质的人单核细胞分离的不同疾病的情况下, 并评估血液中的因素, 可能会提高这种倾向。

视频: Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

科研

免疫与感染

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin injury, inflammation, and/or sensitization. The goal of this study is to describe an epidermal-dermal separation protocol using thermolysin, a proteinase that is active at 4 &deg;C. To illustrate this procedure, Sprague Dawley rats are anesthetized, and right hind paws are injected with carrageenan. Six and twelve hours after injection, rats with inflammation and na&iuml;ve rats are euthanized, and a piece of hind paw, glabrous skin is placed in cold Dulbecco's Modified Eagle Medium. The epidermis is then separated at the basement membrane from the dermis by thermolysin in PBS with calcium chloride. Next, the dermis is secured by microdissection forceps, and the epidermis is gently teased away. Toluidine blue staining of tissue sections show that the epidermis is separated cleanly from the dermis at the basement membrane. All keratinocyte cell layers remain intact, and the epidermal rete ridges along with indentations from dermal papillae are clearly observed. Qualitative and real-time RT-PCR is used to determine nerve growth factor and interleukin-6 expression levels. Western blotting and immunohistochemistry are finally performed to detect amounts of nerve growth factor. This report illustrates that cold thermolysin digestion is an effective method to separate epidermis from dermis for evaluation of mRNA and protein alterations during inflammation.

Presented here is a protocol for the separation of epidermis from dermis to evaluate inflammatory mediator production. Following inflammation, rat hind paw epidermis is separated from the dermis by thermolysin at 4 &#176;C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.

将鼠皮皮和皮米与热解辛分离，以检测部位特异性炎症mRNA和蛋白质

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin ...