Name: screening assays to characterize novel endothelial regulators involved in the inflammatory response
Uploaded: 2017-09-15T13:00:00
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...

<ol>
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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 ...

Research

JoVE Journal

Immunology and Infection

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

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

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

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

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

Use of Interferon-ÃƒÅ½Ã‚Â³ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

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

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

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 ...

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

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 ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

病毒浓度的测定通过空斑试验：用传统的和新型覆盖系统

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

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

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 ...