Name: screening assays to characterize novel endothelial regulators involved in the inflammatory response
Uploaded: 2017-09-15T13:00:00.000+00:00
Duration: 12 min 50 s
Description: Watch this Scientific Journal Video about Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response at JoVE.com

这项工作得到了 Ministerio de Economía y Competitividad (MINECO) 和干杯卡洛斯 III (ISCIII) 的支持 (赠款编号 IERPY 1149/16 至 a.1;MPY 1410/09 到 s Hortelano);由 MINECO 通过基金 de 研究 en 干杯 () (授予数字 PI11.0036 和 PI14.0055 对 s Hortelano)。Herranz 由 ISCIII IERPY 1149/16 支持。

1. 内皮细胞培养 <ol> <li> 组织培养处理板 <ol> <li> 外套100毫米组织培养板, 2.5 毫升明胶溶液 (蒸压, 蒸馏水中的0.1% 明胶) 30 分钟, #176; C; 此可以推断为所需的井格式。抽出明胶溶液, 在组织培养罩中风干. </li>
	</ol> </li> <li> 组织培养条件 <ol> <li> 在生物孵化器内培养 MLEC-04 细胞 (37 和 #176; C, 95% 湿度, 5% CO 2 )。在 Dulbecco 和 #39 的完整培养基中培养细胞; 改良的鹰培养基: 营养混合物 F-12 (DMEM/F-12), 辅以10% 胎牛血清 (FBS) 和100单位/毫升青霉素和100和 #181; 链霉素 (P/毫升). </li>
		<li> 按标准的 Neubauer 室方法计数单元格, 并在10毫升完整介质中将 MLEC-04 细胞添加到100毫米明胶处理的板上, 在低密度 2-11 x 10 4 单元/cm 2 。当细胞到达汇合处时, 拆分1:3 单元格. 
		注意: 通常这在文化中发生了二到三天. </li>
		<li> 通过用10毫升无菌磷酸缓冲盐水 (PBS) 洗涤板来培养细胞, 然后加入2毫升胰蛋白酶-edta 溶液 (0.25% 胰蛋白酶, 5 毫米 edta), 在3和 #176 孵育37分钟;
		<li> 停止胰蛋白酶-EDTA 反应, 并通过添加10毫升完整介质来恢复悬浮细胞。自旋下来的细胞 (300 x g, 5 分钟), 丢弃上清, 重细胞颗粒在完整的媒体和亚文化的适当. </li>
	</ol> </li> </ol> 2。LPS 治疗和调解人 <ol> <li> 将完整介质中的 MLEC-04 单元 sub-confluence 成以下井格式: 7 x 10 5 单元格/井在6井板和 2.5 x 10 5 单元格/井在 96-井板上. </li>
	<li> 在细胞孵化器中孵育 6 h 的培养 (37 和 #176; C, 95% 湿度, 5% CO 2 )。将单元格切换到不完整介质 (DMEM-F12、P/S), 并在夜间 (ON) 离开以同步并减少单元活动. </li>
	<li> 通过在不完整的介质 (30 分钟、37和 #176; C) 中稀释的 (或不含) 药物, 将内皮细胞与 (或不) 有关的物质 (如 DT 10 ) 进行孵化, 从而去除介质和测试新的药理化合物; 添加 1.4 mL/6-井板或0.1496-井板). </li>
	<li> 通过在每种化验中指定的时间内加入 LPS 向孵化培养基 (100 ng/毫升, 最终浓度) 挑战细胞。使用 PBS 作为控件. </li>
</ol> 3。RT qPCR 对活化内皮细胞转录谱的评价 <ol> <li> 在6井培养皿 (7 x 10 5 单元格/井) 中 MLEC-04 种子 sub-confluence。孵育6小时 (37 和 #176; C, 95% 湿度, 5% CO 2 ), 然后进行血清饥饿 (孵育). </li>
	<li> 移除介质并治疗 (或不治疗) 不完全介质中稀释的有兴趣的药物的内皮细胞培养物 (孵育30分钟, 37 和 #176; c); 添加1.4 毫升/6 孔板 (30 分钟, 37 和 #176; c). </li>
	<li> 通过向孵育介质添加 100 ng/mL LPS 来挑战细胞 (如步骤2.3 所述), 并孵育 6 h. 停止反应, 用冷 PBS 冲洗两次, 并在-80 和 #176 中保持板, 直到样品处理. </li>
	<li> rna 提取 <ol> <li> 解冻板并添加1毫升/井 RNA 提取缓冲器 (38% 苯酚和0.8 米胍异硫氰酸酯; 见材料表)。在室温 (RT) 下30分钟, 在1.5 毫升的管子内进行搅拌并收集匀浆. </li>
		<li> 添加200和 #181; L 氯仿和温和搅拌十五年代. 在 RT 和离心机上孵育3分钟 (11600 x g、15 min、4和 #176; C). </li>
		<li> 将水相转移到另1.5 毫升管。通过加入500和 #181, 沉淀 RNA, 然后在 RT, 然后离心 (11600 x g, 4 和 #176; C) 后进行10分钟的孵育. </li>
		<li> 用75% 乙醇通过涡流搅拌然后离心 (7500 x g, 4 和 #176; C) 去除上清液和洗涤颗粒. </li>
		<li> 气干颗粒和溶解 RNA 在25和 #181; 纯 H 2 O 通过孵化10分钟, 在55和 #176; c. 将 RNA 保持在-80 和 #176; c 直到样品处理. </li>
	</ol> </li> <li> 数量和纯度 rna <ol> <li> 通过测量分光光度计中样品在 260 nm 的吸收度来量化 rna 浓度 (参见 材料表 ). </li>
		<li> 测量在 230 nm 和 280 nm, 以确定 RNA 纯度. 
		注: 260/280 的比值表示蛋白质或苯酚污染。接近2的比率是可以接受的。260/230 的比值表明 EDTA、碳水化合物或苯酚污染物。2.0-2.2 之间的值是可接受的. </li>
	</ol> </li> <li> 检查 RNA 完整性 <ol> <li> 运行2和 #181; g 的总 rna 和一个 rna 阶梯上的1.5% 变性琼脂糖凝胶; 凝胶文档系统的可视化 (参见 材料表 ). 
		注: 2:1 的清晰和尖锐的28S 和 18S rRNA 波段的比率表示一个完整的 RNA。部分退化的 RNA 作为一个污点的外观解决. </li>
	</ol> </li> <li> RT-qPCR <ol> <li> 根据标准协议 (参见 材料目录 ) 11 , 从单个搁浅的 RNA 中合成互补 DNA (cDNA). </li>
	</ol> </li> <li> 通过 RT-qPCR <ol> <li> 为每个样本准备反应混合物: 混合2和 #181; l cDNA, 7 和 #181; l 荧光化合物, 300 nm 前引物, 和 300 nm 反向底漆 ( 表 1 ) 在最终体积为13和 #181; L, 并添加到96井反应板 (请参见 材料表 ). </li>
		<li> 用光学粘合剂盖封板, 离心机 (300 x g, 1 分钟) 并在实时 PCR 系统上运行反应 (热启动95和 #176; c 为二十年代, 其次为40个周期:95 和 #176; c 为 3 s 和60和 #176; c 为三十年代) (请参见 材料表 ). </li>
		<li> 使用比较方法 2 和 #916 对结果进行相对定量分析; #916; Ct 与内务管理基因 glyceraldehyde-3-phosphate 脱氢酶 (GAPDH) 或酸性核糖体磷 P0 (36B4) 12.</li>
	</ol> </li> </ol> 4。流式细胞仪评价内皮细胞活化作用 <ol> <li> 按照2节中描述的条件处理 MLEC-04 细胞, 并通过流式细胞仪评估内皮表面蛋白的变化. </li>
	<li> 在步骤1.2.3 中描述的胰蛋白酶-EDTA 法对 6 h LPS 刺激后的细胞进行分离, 用4和 #176 的不完全介质冲洗;
	<li> 使用 Neubauer 室方法计算单元格, 并将 10 x 10 4 单元格放入 u 底 96-井板中。离心机 (300 x g, 5 分钟), 用180和 #176 丢弃上清液; 从上到下的手腕折断, 并迅速恢复到原来的位置. </li>
	<li> 添加50和 #181; 在不完全介质中稀释的选定抗体 (10 和 #181; 最终浓度) 的细胞和孵育 (30 分钟, 4 和 #176; C). </li>
	<li> 将单元格清洗两次 w根据步骤4.3 中所述的程序不完整的介质, 用50和 #181 孵育; L 在10和 #181; 与 FITC 或类似的共轭 (30 min, 4 和 #176; C 在一个黑暗的空间) 的对应的二级抗体的 g/毫升. </li>
	<li> 用不完整的介质冲洗一次, 然后用 PBS 洗涤。用300和 #181 恢复细胞; L PBS 使用1毫升吸管技巧和在细胞仪管中放置. </li>
	<li> 评估流式细胞仪系统中的样本 (请参阅 材料表 )。通过向前和侧面散射参数调整单元格的数量。门的人口利益。根据同种控制的细胞的负控制调节闸门。分析结果为阳性细胞或平均荧光强度的百分比 8 . </li>
</ol> 5。用免疫印迹法评价内皮细胞活化作用 <ol> <li> 在6井培养皿 (7 x 10 5 细胞/井) 中 sub-confluence 内皮细胞, 并按2节所述处理 LPS。通过下面的印迹协议分析炎症蛋白表达. </li>
	<li> 停止 lps 刺激在不同的时间点, 以阐明不同方面的内皮反应: <ol> <li> 确定炎症蛋白剖面后 6 h 的 lps 刺激. </li>
		<li> 每15分钟通过60分钟的时间确定单元格信号. </li>
	</ol> </li> <li> 在这两种情况下, 通过用冷 PBS 冲洗两次以停止反应, 并将样品存储在-80 和 #176; C 直到样品处理. </li>
	<li> 溶解单元格和蛋白质提取 <ol> <li> 添加200和 #181; 每井溶解缓冲液 (1% 非离子表面活性剂, 10 毫米三盐酸, 1mM EDTA, 150 毫米氯化钠, 30 毫米焦磷酸钠, 50 毫米氟化钠, 2.1 毫米钠钒pH 值 7.6, 辅以蛋白酶抑制剂鸡尾酒) (见材料表). </li>
		<li> 孵育15分钟, 在4和 #176; C 在搅拌下, 用吸管尖刮井, 并在1.5 毫升管中收集匀浆. </li>
		<li> 离心管在 11600 x g 在4和 #176; C. </li>
		<li> 在-80 和 #176 中将清液储存在清洁的1.5 毫升管中, 直到处理. </li>
	</ol> </li> <li> 按照标准协议 (参见材料表) 13 来测量二辛可宁酸含量的蛋白质浓度. </li>
	<li> 解决30和 #181; 通过标准技术0.1% 十二烷基硫酸钠、10% 聚丙烯酰胺凝胶电泳 (SDS 页) 14 对每个样品的总蛋白裂解 g。使用10毫米和 #946 的样品, 基 (还原条件) 或无 (还原条件), 取决于用于检测感兴趣的蛋白质的抗体. </li>
	<li> 将分离的蛋白质从聚丙烯酰胺凝胶转移到聚偏氟 (PVDF) 转移膜, 其0.45 和 #181; m 孔径采用标准程序. </li>
	<li> 在迁移后, 用 pbs 两次冲洗膜, 0.1% polysorbate-20 (pbs t) 和阻断不结合点, 加入20毫升 2% BSA 稀释在 pbs t 中检测磷酸或5% 脱脂奶粉中的总蛋白, 在搅拌下 (90 分钟,RT). </li>
	<li> 将所选抗体稀释在适当的阻塞缓冲液的10毫升中, 在推荐浓度下, 并在搅拌下孵育膜 (上、4和 #176; C)。或者, 将膜放在密封的塑料袋中, 用5毫升稀释的抗体. </li>
	<li> 第二天, 冲洗膜三次 (过量 PBS t, 15 分钟, RT). </li>
	<li> 在适当的阻断缓冲液中, 用10毫升与辣根过氧化物酶 (HRP) 稀释的对应二级抗体, 在搅拌 (30 分钟, RT) 下孵育膜. </li>
	<li> 在搅拌下冲洗三次膜 (过量 PBS-T, 15 分钟, RT). </li>
	<li> 孵育的膜为1分钟, 0.1 毫升/厘米 2 的过氧化物酶基质增强化学发光 (ECL)。将排水膜放在两个透明塑料板之间, 并将其插入到化学发光检测系统中, 其中包括带电耦合的器件摄像机 (CCD). 
	<ol> <li> 使用 CCD 摄像机检测信号及其软件, 以累计程序记录图像 (在每三十年代曝光时图像显示累计信号, 总时间为15分钟). </li>
	</ol> </li> <li> 根据用户指南 15 中所述的协议, 使用 ImageJ 软件量化密度。
	<ol> <li> 打开 ImageJ 软件中的示例, 并使用矩形选择工具选择感兴趣的带区. </li>
		<li> 选择作为第一条车道, 并按绘图车道获取剖面图. </li>
		<li> 用 straight-line 选区分隔峰值区域. </li>
		<li> 通过单击每个峰值的内侧来测量每个波段的大小. </li>
		<li> 表示有关加载蛋白质控制 (和 #946;-肌动蛋白, 蛋白, 等的结果 ). </li>
	</ol> </li> </ol> 6。粘附测定白细胞活化内皮因子的评价 <ol> <li> 获取内皮条件介质。
	<ol> <li> 治疗2节中所示的 MLEC-04 细胞, 并在 24 h 刺激与 LPS (100 ng/毫升) 后收集培养上清液. </li>
		<li> 从处理过的 MLEC-04 细胞和离心机收集被适应的媒介 (600 x g)。将上清液保持在0.5 毫升等分在-80 和 #176; C 直到使用. </li>
	</ol> </li> <li> 白细胞粘附检测 <ol> <li> 将96个井板涂上50和 #181; 在 PBS 中稀释的选择的胞外基质蛋白的 L/井 (胶原蛋白, 在0.1 米醋酸中稀释): 纤维粘连蛋白 (1-10 和 #181; g/毫升), 层粘连蛋白 (1-10 和 #181; g/毫升) 和胶原 i 型 (10-40 和 #181; g/毫升)。4和 #176 离开, C. </li>
		<li> 丢弃涂布介质, 并在150和 #181 的水井上阻断不结合位点, 1% BSA 热灭活 (1 分钟, 100 和 #176; C), 在 RT 上为90分钟. </li>
		<li> 用 PBS 冲洗两次井, 并添加100和 #181; L 以前收集的内皮条件介质 (6.1 节). 
		注: 板已准备好进行附着力测定. </li>
		<li> 在生物孵化器中培养小鼠单核-巨噬细胞系 J774 (37 #176; C, 95% 湿度, 5% CO 2 ) 与罗斯威尔公园纪念研究所媒体 (RPMI), 10% FBS, 1% P/秒. 
		注: J774 细胞在悬浮中生长. </li>
		<li> 收集 J774 细胞到15毫升管和离心机 (300 x g, 5 min)。用10毫升无血清培养基丢弃上清和重细胞颗粒。在 Neubauer 室中计数细胞。离心细胞 (300 x g, 5 min), 丢弃上清和重在无血清培养基中的细胞在 15 x 10 4 J774 细胞每50和 #181; L 媒体. </li>
		<li> 将 15 x 10 4 J774 单元格添加到每个井中50和 #181; L 无血清培养基, 在15和 #176 孵育4分钟; c 在1和 #176; c 在 CO 37 细胞孵化器. </li>
		<li> 冲洗两次井, 轻轻地加入温水 PBS; 离心机 (300 x g, 5 分钟), 用180和 #176 丢弃上清液; 手腕从上到下, 快速恢复到原来的位置。修复附加的单元格在 PBS 和孵育中加入100和 #181; 4% 甲醛 (粉煤灰) 的 L/井 (10 分钟, RT). </li>
		<li> 将介质轻轻地丢弃, 并在 PBS 中用2% 甲醇 permeabilize 电池2分钟. 将介质轻轻丢弃, 并通过添加50和 #181 对电池进行着色; 九十年代在 rt 中20% 甲醇中的0.5% 结晶紫的 L/井. </li>
		<li> 用大量的自来水冲洗盘子, 丢弃多余的液体, 使其风干。用数码相机将附着的细胞报告给光显微镜. </li>
		<li> 通过添加100和 #181 稀释细胞染色; 0.1 米柠檬酸钠, 50% 乙醇。测量 545 nm 的吸光度, 并将其结果表示为任意单位的吸光度或附着力百分比, 方法是将100% 作为附着在高配体浓度的井中的细胞黏附力 </li> </ol> </li> </ol> 7。白细胞-内皮细胞共粘法检测内皮细胞活化 <ol> <li> 在2节所述的 96-井板上处理 MLEC-04 单元, 并与 LPS 孵育 (6 h、37和 #176; C). </li>
	<li> 荧光标签 J774 细胞与羧基琥珀酰亚胺酯 (CSFE). 
	<ol> <li> 按照6.2.5 中的过程清洗 PBS 中的 J774 单元格。通过 Neubauer 室法和重在 PBS 中的 1 x 10 6 单元/mL 计数单元格, 0.1% BSA. </li>
		<li> 用 CFSE (5 #181; M, 最终浓度) 在37和 #176 孵育细胞20分钟; c. 添加5毫升不完全培养基和孵育 (5 分钟, 4 和 #176; c). </li>
		<li> 使用不完整介质清洗一次, 如6.2.5 中所述, 重在 15 x 10 4 cells/100 和 #181; L 并进行 co-adhesion 检测. </li>
	</ol> </li> <li> 在内皮治疗后, 用不完整的介质冲洗三次井。将 CFSE-J774 单元格 (15 x 10 4 cells/100 #181; L) 添加到每个内皮涂层井。在4和 #176 中孵育板材10分钟; c 依次为60分钟, 在37和 #176; c. </li>
	<li> 轻轻地清洗水井, 如6.2.7 中所述, 使用加温 PBS, 并报告 J774 粘附到内皮层的方法有以下两种: <ol> <li> 溶解 0.1 M 中的细胞, 8.8, 1% SDS (100 和 #181; L/井) 并测量CFSE-J774 信号由荧光 (励磁或放射 = 492 nm/517 毫微米)。将结果表示为任意单位的荧光强度或与阳性对照的粘附百分比 (来自每个井的总细胞的信号). </li>
		<li> 修复4% 粉煤灰中的细胞, 通过可视化荧光显微镜 (激发/发射 = 492 nm/517 nm) 将 CFSE-J774 细胞的附着报告给内皮. </li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

这种内皮细胞的协议描述了一种逐步的技术, 建立了探索新的机制参与调节炎症反应的基础。这些方法是基于对 LPS 刺激的内皮活性的研究, 并评估在炎症反应过程中所涉及的白细胞招募的关键步骤, 特别是: 内皮细胞因子释放, 内皮黏附分子表达和白细胞粘附到血管层。一旦血管内皮细胞的参数建立, 系统可以寻找新的化合物参与调节内皮功能, 从而, 炎症进展。这些监管药物可能对医药市场有潜在?...

炎症是对感染性药物的有益的生物学反应, 其主要目的是消除病原体, 修复受损组织。在某些情况下, 如慢性感染或自身免疫性疾病, 炎症无法解决。相反, 有一个异常的反应与持续的白细胞浸润, 导致长期免疫反应, 导致组织损伤, 纤维化, 功能丧失, 和整体, 残疾, 在某些情况下死亡的病人。这些人类疾病, 归类为炎症性疾病, 都涉及血管的控制白细胞外渗1,2。
内皮细胞通过控制白细胞的贩运在调节炎症反应中起着根本性的作用。当内皮层暴露在炎症介质, 如 LPS, 休眠内皮激活和表达炎细胞因子 (Cxcl10, Cxcl5, Cxcl1,等) 和黏附分子 (e-选择素, VCAM-1 和 ICAM-1), 有利于对感染部位进行循环白细胞的招募。由释放的细胞因子引导的白细胞, 然后调解滚动和互动的内皮层通过相应的胶粘剂对应: PSGL-1 的选择, α4β1整合素 VCAM-1, 和αLβ2整合素 ICAM-1。最后, 白细胞在血管中迁移到炎症的焦点3。
内皮细胞在调节炎症反应中所起的重要作用已经在基因修饰的小鼠身上得到证实, 它只在内皮细胞膜上表达 LPS 受体, 即 toll 样受体 4 (TLR4)。这些 endothelial-TLR4 动物能够对 LPS 介导的炎症反应和检测细菌接种后产生的感染, 从而达到与野生型小鼠一样的感染分辨率和生存率4,5。
对于内皮调节炎症反应通路, 已假设, 在某些阶段的白细胞-内皮相互作用的抑制将导致反式内皮细胞的迁移减少, 并有更好的预后炎症相关疾病。事实上, 一些针对内皮细胞活化和白细胞-内皮相互作用的策略被设计成阻碍免疫的外渗作为治疗炎症性疾病的方法6,7。
在本报告中, 我们描述了一个完整的组的体外技术, 以充分表征内皮活性响应炎症刺激 LPS 及其作用的白细胞活化和粘附到血管层。该手稿中使用的内皮细胞模型是小鼠肺内皮细胞系 (MLEC-04), 如 Hortelano et al.所描述的。8. 在文献中验证了 MLEC-04 细胞系是研究内皮细胞活化的适当系统9,10。根据研究兴趣, 这些方法可以很容易地推断出任何内皮或白细胞系统和炎症剖面。一旦确定了所选条件下的内皮参数, 系统就可以对新的药物进行实验, 以评价血管活化。在这种炎症的背景下, 用复利测定的内皮细胞可以与细胞的控制条件相比较, 任何结果的差异都可能使药物的预后与炎症的发展和进展有关。最后, 我们提出了一个相关的系统来表征新的药物靶向内皮细胞, 这可以影响设计的新型血管特异治疗炎症相关疾病。

<table><tbody><tr><td>Gelatin</td><td>Sigma</td><td>G9391</td><td></td></tr><tr><td>DMEM-F12</td><td>Lonza</td><td>BE12-719F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>A4503</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Lonza</td><td>DE17-602E</td><td></td></tr><tr><td>Trypsine</td><td>Lonza</td><td>BE17-160E</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>ED2SS</td><td></td></tr><tr><td>LPS</td><td>Sigma</td><td>L2880</td><td></td></tr><tr><td>Trizol</td><td>Sigma</td><td>T9424</td><td>RNA extraction buffer</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>33539</td><td></td></tr><tr><td>Ethanol absoluto</td><td>Panreac</td><td>1,310,861,612</td><td></td></tr><tr><td>Pure H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Qiagen</td><td>1017979</td><td>RNAse free</td></tr><tr><td>Agarose</td><td>Pronadisa</td><td>8020</td><td></td></tr><tr><td>Stain for agarose gels</td><td>Invitrogen</td><td>s33102</td><td></td></tr><tr><td>SuperScript III First-Strand Synth</td><td>Invitrogen</td><td>18080051</td><td>Reagents for RT-PCR</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>Applied Biosystems</td><td>4385610</td><td>Fluorescent stain for qPCR</td></tr><tr><td>MicroAmp Fast Optical 96-Well</td><td>Applied Biosystems</td><td>4346906</td><td>Plates for qPCR</td></tr><tr><td>U-bottom 96 well plates</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Cytometry tubes</td><td>Falcon</td><td>352054</td><td></td></tr><tr><td>TX100</td><td>Panreac</td><td>212314</td><td>Non-ionic surfactant</td></tr><tr><td>Tris-HCl</td><td>Panreac</td><td>1,319,401,211</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>1,064,041,000</td><td></td></tr><tr><td>Sodium pyrophosphate</td><td>Sigma</td><td>221368</td><td></td></tr><tr><td>Sodium fluoride</td><td>Sigma</td><td>S7920</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>sigma</td><td>13721-39-6</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>sigma</td><td>P8340</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Pierce</td><td>23225</td><td>Reagents for bicinchoninic acid assay</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>merck</td><td>805,740</td><td></td></tr><tr><td>PVDF Transfer Membrane, 0.45 &amp;micro;m</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Tween-20</td><td>Panreac</td><td>1,623,121,611</td><td>Polysorbate 20</td></tr><tr><td>PBS</td><td>Lonza</td><td>BE17-515Q</td><td></td></tr><tr><td>ECL</td><td>Millipore</td><td>WBKLS0500</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Collagen type I</td><td>Sigma</td><td>c8919</td><td></td></tr><tr><td>Acetic acid</td><td>Panreac</td><td>1,310,081,611</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Methanol</td><td>Panreac</td><td>1,310,911,612</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma</td><td>HT90132</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>C7254</td><td></td></tr><tr><td>Ethanol 96%</td><td>Panreac</td><td>1,410,851,212</td><td></td></tr><tr><td>CFSE</td><td>Sigma</td><td>21888</td><td></td></tr><tr><td>RPMI</td><td>Lonza</td><td>BE12-115F</td><td></td></tr><tr><td>SDS</td><td>Bio-Rad</td><td>161-0418</td><td></td></tr><tr><td>Infinite M200</td><td>Tecan</td><td>M200</td><td>Multi mode microplate reader</td></tr><tr><td>Gel Doc 2000</td><td>Bio-Rad</td><td>2000</td><td>Gel documentation system</td></tr><tr><td>StepOnePlus</td><td>Applied Biosystems</td><td>StepOnePlus</td><td>qPCR system</td></tr><tr><td>MACSQuant Analyzer 10</td><td>Miltenyi Biotec</td><td>Analyzer 10</td><td>Cytometry equipment</td></tr><tr><td>ChemiDoc MP</td><td>Bio-Rad</td><td>MP</td><td>Chemiluminescence detection system</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PECAM-1</td><td>BD Biosciences</td><td>553370</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>ICAM-2</td><td>Biolegend</td><td>1054602</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>E-selectin</td><td>BD Biosciences</td><td>553749</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>VCAM-1</td><td>BD Biosciences</td><td>553330</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>ICAM-1</td><td>Becton Dickinson</td><td>553250</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>anti-rat IgG-FITC</td><td>Jackson Immuno Research</td><td>112-095-006</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>anti armenian hamster-FITC</td><td>Jackson Immuno Research</td><td>127-095-160</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>Rat IgG isotyope control</td><td>Invitrogen</td><td>10700</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>Armenian hamster IgG isotype control</td><td>Invitrogen</td><td>PA5-33220</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>P-I&amp;kappa;&amp;Beta;-&amp;alpha;</td><td>Cell Signaling</td><td>2859</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>&amp;beta;-Actin</td><td>Sigma</td><td>A5441</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>P-ERK</td><td>Cell Signaling</td><td>9101</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>anti-mouse HRP</td><td>GE Healthcare</td><td>LNXA931/AE</td><td>Use at 1:10,000</td></tr><tr><td>anti-rabbit HRP</td><td>GE Healthcare</td><td>LNA934V/AG</td><td>Use at 1:10,000</td></tr><tr><td>anti-rat HRP</td><td>Santa Cruz</td><td>Sc-3823</td><td>Use at 1:10,000</td></tr></tbody></table>

screening assays to characterize novel endothelial regulators involved in the inflammatory response

内皮层是必不可少的, 以维持体内的稳态, 通过控制许多不同的功能。调节炎症反应的内皮层是至关重要的有效打击有害的投入和援助, 恢复受损地区。当内皮细胞暴露在炎症环境中, 如 gram-negative 细菌膜的外成分, 脂多糖 (LPS), 它们表达可溶性炎细胞因子, 如 Ccl5, Cxcl1 和 Cxcl10, 并触发循环白细胞的活化。此外, 黏附分子 e-选择素、VCAM-1 和 ICAM-1 在内皮表面的表达能使活化白细胞与内皮层相互作用和黏附, 并最终向发炎的组织渗出。在这种情况下, 内皮功能必须受到严格的调节, 因为过量或有缺陷的活化在白细胞的招募可能导致炎症相关的疾病。由于许多这些疾病没有有效的治疗, 新的战略, 重点在血管层必须研究。我们建议全面的化验, 是有用的, 以寻找新的内皮调节剂, 改变白细胞功能。我们通过使用特定的表达靶点 (如细胞因子、趋化因子和黏附分子) 来分析内皮细胞活化作用, 包括: real-time 定量聚合酶链反应 (qPCR), 印迹, 流式细胞仪和粘附化验。这些方法可以确定炎症环境中的内皮功能, 对进行筛选化验以表征新的血管内皮炎症调节剂是非常有用的, 这对于设计治疗策略具有潜在的价值。

qPCR 对 LPS 诱导的内皮细胞活化作用的评价
在6小时内, 用100毫升 LPS 刺激血清 MLEC-04 细胞, 通过比较活化标记物的表达与静止状态的 qPCR, 对内皮基因表达进行评价。如图 1A所示, LPS 孵育的 MLEC-04 细胞诱导了在炎症反应 (e-选择素、VCAM-1 和 ICAM-1) 中参与白细胞招募的选择性黏附分子的 mRNA 表达。PECAM-1 被用来作?...

Watch this Scientific Journal Video about Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response at JoVE.com

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

血管内皮严密控制白细胞的招募。不充分的白细胞外渗导致人类炎症性疾病。因此, 寻找新的调节元素内皮活化是必要的设计改进治疗炎症性疾病。在这里, 我们描述了一个全面的方法来表征新的内皮调节剂, 可以改变白细胞贩运在炎症。

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

screening-assays-to-characterize-novel-endothelial-regulators

Research

JoVE Journal

Immunology and Infection

6.5K 次观看.  Institute of Health Carlos III. 血管内皮严密控制白细胞的招募。不充分的白细胞外渗导致人类炎症性疾病。因此, 寻找新的调节元素内皮活化是必要的设计改进治疗炎症性疾病。在这里, 我们描述了一个全面的方法来表征新的内皮调节剂, 可以改变白细胞贩运在炎症。

视频: Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

评估与远藤的PAT 2000内皮血管舒张功能

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

科研

医学

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

片上内皮炎症表型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

生物工程

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

促进药物发现：斑马鱼在一个自动化的高含量检测炎症

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

免疫与感染

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

发育生物学

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

用于炎症反应的新内皮调节因子的筛选测定

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