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>

<ol>
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SuperScript&#174; III First-Strand Synthesis System for RT-PCR Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/manuals/superscriptIIIfirststrand_pps.pdf">https://tools.thermofisher.com/content/sfs/manuals/superscriptIIIfirststrand_pps.pdf</a> (2013)</li><li>Livak, K. J., Schmittge, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>. Pierce BCA Protein Assay Kit Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf">https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf</a> (2017)</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laemmli-SDS-PAGE.">Laemmli-SDS-PAGE.</a> BIO-PROTOCOL. 1 (11), (2011).</li><li>. ImageJ User Guide Available from: <a target="_blank" href="https://imagej.nih.gov/ij/docs/guide/user-guide-USbooklet.pdf">https://imagej.nih.gov/ij/docs/guide/user-guide-USbooklet.pdf</a> (2017)</li><li>Hohsfield, L. 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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 border="0" cellpadding="0" cellspacing="0" width="820">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gelatin</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:116px;">G9391</td>
			<td style="width:256px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM-F12</td>
			<td>Lonza</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin</td>
			<td>Lonza</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsine</td>
			<td>Lonza</td>
			<td>BE17-160E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma</td>
			<td>ED2SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>Sigma</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td>Sigma</td>
			<td>T9424</td>
			<td>RNA extraction buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td>Sigma</td>
			<td>33539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol absoluto</td>
			<td>Panreac</td>
			<td>1,310,861,612</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Pure H2O</td>
			<td>Qiagen</td>
			<td>1017979</td>
			<td>RNAse free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Pronadisa</td>
			<td>8020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stain for agarose gels</td>
			<td>Invitrogen</td>
			<td>s33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III First-Strand Synth</td>
			<td>Invitrogen</td>
			<td height="21" style="height:21px;width:116px;">18080051</td>
			<td>Reagents for RT-PCR</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Fast SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td align="left">4385610</td>
			<td>Fluorescent stain for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroAmp Fast Optical 96-Well</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td>Plates for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-bottom 96 well plates</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytometry tubes</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TX100</td>
			<td>Panreac</td>
			<td>212314</td>
			<td>Non-ionic surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Panreac</td>
			<td>1,319,401,211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Merck</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyrophosphate</td>
			<td>Sigma</td>
			<td>221368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Sigma</td>
			<td>S7920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium orthovanadate</td>
			<td>sigma</td>
			<td>13721-39-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor cocktail</td>
			<td>sigma</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Pierce BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>Reagents for bicinchoninic acid assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td>merck</td>
			<td align="left">805,740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">PVDF Transfer Membrane, 0.45 &#181;m</td>
			<td>Thermo Scientific</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Panreac</td>
			<td align="left">1,623,121,611</td>
			<td>Polysorbate 20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Lonza</td>
			<td>BE17-515Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECL</td>
			<td>Millipore</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen type I</td>
			<td>Sigma</td>
			<td>c8919</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid</td>
			<td>Panreac</td>
			<td>1,310,081,611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Panreac</td>
			<td>1,310,911,612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Crystal violet</td>
			<td>Sigma</td>
			<td>HT90132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate</td>
			<td>Sigma</td>
			<td>C7254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 96%</td>
			<td>Panreac</td>
			<td>1,410,851,212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFSE</td>
			<td>Sigma</td>
			<td>21888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Lonza</td>
			<td>BE12-115F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>Bio-Rad</td>
			<td>161-0418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infinite M200</td>
			<td>Tecan</td>
			<td>M200</td>
			<td>Multi mode microplate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel Doc 2000</td>
			<td>Bio-Rad</td>
			<td>2000</td>
			<td>Gel documentation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StepOnePlus</td>
			<td>Applied Biosystems</td>
			<td>StepOnePlus</td>
			<td>qPCR system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSQuant Analyzer 10</td>
			<td>Miltenyi Biotec</td>
			<td>Analyzer 10</td>
			<td>Cytometry equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChemiDoc MP</td>
			<td>Bio-Rad</td>
			<td>MP</td>
			<td>Chemiluminescence detection system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PECAM-1</td>
			<td>BD Biosciences</td>
			<td>553370</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ICAM-2</td>
			<td>Biolegend</td>
			<td>1054602</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">E-selectin</td>
			<td>BD Biosciences</td>
			<td>553749</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">VCAM-1</td>
			<td>BD Biosciences</td>
			<td>553330</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ICAM-1</td>
			<td>Becton Dickinson</td>
			<td>553250</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-rat IgG-FITC</td>
			<td>Jackson Immuno Research</td>
			<td>112-095-006</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti armenian hamster-FITC</td>
			<td>Jackson Immuno Research</td>
			<td>127-095-160</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG isotyope control</td>
			<td>Invitrogen</td>
			<td style="width:116px;">10700</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Armenian hamster IgG isotype control</td>
			<td>Invitrogen</td>
			<td>PA5-33220</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P-I&#954;&#914;-&#945;</td>
			<td>Cell Signaling</td>
			<td>2859</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Actin</td>
			<td>Sigma</td>
			<td>A5441</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P-ERK</td>
			<td>Cell Signaling</td>
			<td>9101</td>
			<td>Use at 10 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse HRP</td>
			<td>GE Healthcare</td>
			<td>LNXA931/AE</td>
			<td>Use at 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-rabbit HRP</td>
			<td>GE Healthcare</td>
			<td>LNA934V/AG</td>
			<td>Use at 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-rat HRP</td>
			<td>Santa Cruz</td>
			<td>Sc-3823</td>
			<td>Use at 1:10000</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 ...

The overall goal of this screening assay is to characterize novel endothelial regulators involved in the inflammatory response of interest for designing new therapeutic strategies against inflammatory disorders.This method can help answer key questions in the pathology field such as defining novel endothelial-targeting drugs able to attenuate inflammatory-related diseases.The main advantage of this endothelial protocol is that describes assay technology that establishes the basis for exploring novel mechanisms involved in the regulation of the inflammatory response.Demonstrating this methodology will be Maria Angeles Higueras, a post-doc from our lab and Sandra Herranz, a technician from our lab.To begin, culture MLEC-04 cells in complete DMEM F12 medium supplemented with 10%FBS and Pen-Strep.Incubate the cells in a biological incubator at 37

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

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Immunology and Infection

6.5K 次观看.  Institute of Health Carlos III. The overall goal of this screening assay is to characterize novel endothelial regulators involved in the inflammatory response of interest for designing new therapeutic strategies against inflammatory disorders.This method can help answer key questions in the pathology field such as defining novel endothelial-targeting drugs able to attenuate inflammatory-related diseases.The main advantage of this endothelial protocol is that describes assay technology that establishes the basis for explorin...

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

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学：生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

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 ...