该项目的筹资提供了由美国癌症协会的研究学者奖（06 - 024 - 01 - CSM的查尔斯J Dimitroff）;国家机构的健康/美国国家癌症研究所资助（RO1CA118124查尔斯J. Dimitroff）;和国家卫生研究所/国家关节炎，肌肉骨骼及皮肤疾病授予的研究院（P30 AR042689托马斯博士S. Kupper;布里格姆与妇女医院，马萨诸塞州波士顿）。

1。 HBMEC制备单层（HBMEC - 60细胞）在培养皿流室<ol><li>大衣35 x 10mm的组织培养皿在372毫升20μg/ml纤维连接蛋白（在PBS）在4℃过夜，或为3小时° C。 </li><li>吸菜纤维连接蛋白的解决方案，并添加 5 HBMEC 1.5 X10 - 60细胞[中199 HEPES及谷氨酰胺，10％胎牛血清，10％的人血清，5个单位/ ml肝素，1ng/ml重组人成纤维细胞生长因子， 1％青霉素链霉素]的菜，让他们成长到90％汇合。 （2天） </li><li>吸生长介质，并上调E -选择素的表达，增加与50ng/mL的IL -1β的新鲜培养基为4-6小时。细胞单层现在准备滚动检测。要阻止E -选择素的功能，中抗人E -选择素单克隆抗体（克隆BBIG - E4（5D11））可以添加在37〜1小时，在20μg/ml° C。 </li></ol> 2。人体造血祖流室KG1a细胞的制备<ol><li>人体造血祖KG1a细胞生长至汇合（1 × 10 6细胞/ ml）的RPMI - 1640谷氨酰胺和10％FBS / 1％的青霉素，链霉素和吸管直接从烧瓶在流动腔实验中使用。 </li><li>细胞数计算hemacytometer使用，然后在1 × 10 6细胞/ ml的细胞重新悬浮在10MM HEPES（H / H缓冲液）和2mm氯化钙2的HBSS </li><li>存储在冰块，直到在实验中使用的细胞。 </li></ol> 3。制备显微镜，平行板流动腔和注射泵（图1） <img src="/files/ftp_upload/old/1009_2009_1_26_7/1009_figure1.png" alt="图1" width="600" height="409" /> 图1平行板流动腔分析的插图。倒置显微镜，哈佛注射泵，计算机/摄像机和平行板流动腔放置在效率和可再生的细胞分析的最佳安排。 <ol><li>倒置显微镜和微量注射泵的电源，并打开电源和光源选择在显微镜10X目的。 </li><li>在持有人与H / H和2mm 氯化钙 50毫升锥形管和填充。 </li><li>下面的测试50ml的锥形管的管持有人将5毫升塑料试管</li><li>放置在60ml的注射器注射泵（务必注射器的柱塞和身体都担保）。 </li><li>将3路停止公鸡结束60ML注射器5ml注射器和附加3路停止公鸡。 </li><li>广场平行流商会仪器显微镜阶段（GlycoTech公司）与输入管的权利，输出管的左侧和真空集热管的背部。 </li><li>将真空管真空和空气阀打开，让平行板器具坚持阶段。 （如果流室的真空裙是气密试验。）关闭气阀。 </li><li>将输出管行（显微镜左）3路停止公鸡。仪器正准备投放HBMEC - 60单层板。 </li><li>广场HBMEC - 60单层板并将其放置在显微镜中心。 </li><li>打开真空和位置以上的单层板在中心板流动腔装置。 </li><li>轻轻地降低到HBMEC - 60单层板流室装置，并允许流室装置吸。 （应该有逃逸的空气没有的声音，在这一点上，你可能需要施加压力，以商会压缩橡胶垫片。） </li><li>一旦真空已经成立，地方输入管（右）含H / H 2mm的氯化钙2到50毫升锥形管。 </li><li>打开三路停止公鸡，让流“到60ml的注射器和地方泵泵模式。” </li><li>要删除不解除对板细胞的输入/输出线中的空气，使用注射泵2mL/minute设置，以填补进管，然后迅速设置为0.5毫升/分钟之前的流体达到仪器。当务之急是总理此设立小心，避免气泡，将取消单层板。 </li><li>一旦液体被视为迁入60毫升注射器，流室，注射泵已准备使用底漆和流动室。此设置将重复每个单元格输入运行和需要为每个新盘。 </li></ol> 4。捕获使用NIS元素的白细胞滚动事件<ol><li>打开桌面上的NIS元素。 </li><li>按“播放”图标实时图像。自动曝光，将让你看到的板现在更容易，是重要的重点。 </li><li>确定，重点是正确的单层细胞显微镜。 </li><li>一旦集中，重新设置曝光，以1帧/秒，并调整显微镜的光。图像应出现在电脑上，不再由于微光显微镜取景器中可见。光直方图然后可以广告justed删除背景曝光，或提高细胞的清晰度。 </li><li> 2X2 bin或现场斌点击获取图片，录像拍摄的理想选择</li><li>点击菜单栏上的“宏”，并选择“写端口”，端口选择COM3“，”和“开放”。 （这将打开的端口，以配合注射泵。 </li><li>移液器1ML KG1a细胞悬液到附近的输入管行5毫升试管，然后将输入管行试管底部。 </li><li>转到菜单栏，点击“捕获”，“时间推移的收购。” </li><li>会弹出一个新的菜单。选择协议持续210秒，这是HBMEC - 60细胞上滚动的程序的长度。 （编程的每一个阶段是拍照每秒钟泵的协议的期限，这也是成立COM3端口，启动水泵，通过发送命令。）如果中断时间推移，泵不会停止自身必须复位。按一下“取消”返回到实时图像。 </li><li>根据说明书设置注射泵“计划模式”。 （编程模式允许特定的流速（即剪应力水平）传授内室和KG1a细胞间的相互作用决定在HBMEC - 60细胞单层的手工编程。）滚动HBMEC细胞对KG1a细胞的设置如下：4.2达因厘米2 / 45秒，0.3达因/厘米2 60秒，并逐步增加每15秒最多4.2达因/厘米2 </li><li>在会议厅内的壁面切应力（ 宽街）按下列公式计算：ST =6μQ/ A 2 B，其中μ是媒体的估计粘度（0.0076 P），Q是在ML的体积流量/ S，A通道的高度，以厘米（垫片的厚度）（0.03厘米），和B是在厘米（0.5厘米）的通道宽度。流量调节注射泵的使用程序模式。 </li><li>显微镜和计算机现正准备捕捉延时拍摄。按“开始运行”，在计算机上开始采集数据。程序运行时，请务必将媒体添加到包含KG1a细胞的试管中，以保持充满介质的输入/输出管（确保没有气泡）。 </li></ol> 5。终端Sialylation和E -选择素介导的KG1a细胞的表面糖蛋白依赖性。轧件（图2 5.1-5.5视频剪辑） <ol><li>含量的KG1a滚动细胞无刺激HBMEC - 60细胞（阴性对照）KG1a细胞注入到流室超过生活非IL -1β刺激的HBMEC - 60如上所述。 </li><li>测定KG1a细胞滚动HBMEC - 60与IL -1β前处理的细胞（E -选择依赖控制）KG1a细胞注入到流室超过现场的IL -1β刺激的HBMEC - 60如上所述。 </li><li> KG1a细胞测定HBMEC - 60细胞的轧前处理的IL -1β，然后中和抗人E -选择素（E -选择依赖控制 ）单克隆抗体。KG1a细胞准备和在实况流室注入IL - 1β刺激的HBMEC - 60如上所述，中抗人E -选择素单克隆抗体的预处理。 </li><li>唾液酸酶的消化KG1a滚动细胞测定IL -1β刺激的HBMEC - 60。 0.1U/ml霍乱弧菌神经氨酸酶为1小时37℃预处理KG1a细胞的制备和注入流动室以上住白细胞介素-1β刺激的HBMEC - 60如上所述。 </li><li>蛋白酶消化KG1a滚动细胞测定IL -1β刺激的HBMEC - 60。 KG1a细胞与蛋白酶处理，0.2U/ml菠萝蛋白酶在37 ° C为1小时，准备和注入流动腔超过实时的IL -1β刺激HBMEC - 60如上所述。 </li></ol> 6。 NIS元素拍摄的KG1a细胞KG1a细胞的图形滚动数据分析<ol><li>时间序列收购后，保存数据。 </li><li>转到菜单栏上的“测量”选项卡，选择“定义的门槛。”移动所需的细胞，使红色的酒吧。选择“所有图像”，那么“好”。整个时间推移的顺序，现在应该修改可见红色的滚动细胞。 </li><li>转到“办法”，并选择“限制”。 （限制地允许用户排除阈值的对象，并不代表滚动细胞，这主要是由HBMEC - 60单层创建的背景。）选择“区”，输入最小值和最大值滚动细胞领域。重复这一步“伸长”和“圆”。 </li><li>选择“测量”，并选择“创建二进制使用限制”，并设定限制值HBMEC - 60细胞单层优化NIS元素滚动细胞的软件识别。 </li><li>回到“测量”，选择“现场数据”，然后选择“复位数据”。关闭此菜单。 </li><li> “办法”和SElect“扫描领域”。 </li><li>打开“测量”，“现场数据”，选择“数量或对象”，然后选择“各个领域”，将数据导出到Microsoft Excel。 </li><li>选择“清除数据”，以准备下一个数据收集NIS元素细胞采集。 </li></ol>结果： <img src="/files/ftp_upload/old/1009_2009_1_26_8/1009_figure3.png" alt="图3" width="600" height="382" /> 图3平行平板流动商会分析，E -选择约束力的KG1a细胞的活动。唾液酸酶或蛋白酶处理的KG1a细胞上滚动HBMEC - 60与IL -1β前处理的细胞单层效率进行了分析。负控制轧制实验还进行了分析KG1a细胞对IL -1β刺激的HBMEC -60细胞或滚动的IL -1β刺激的HBMEC - 60细胞中和抗人E -选择素单克隆抗体（5D11）治疗。细胞滚动频率分别为一式三份，并与NIS元素软件分析。 （*统计意义，P &lt;0.001） 在此视频为基础的报告，我们提供的可视化描述了如何分析白细胞 - 生理剪应力条件下的内皮细胞的相互作用。特别是，我们分析了E -选择素的作用 - E -选择素配体介导白细胞圈养及压延，粘合剂行为必要的承诺，通过整合和透明质酸受体的受体，如中学更稳定的结合活动。我们使用的平行板流动腔倒置显微镜，哈佛注射器泵，视频摄像机和计算机/细胞采集硬件使用改编，进行实验，其中E -选择素配体+ KG1a细胞是我们的白细胞模型和HBMEC细胞使用（HBMEC - 60）与促炎性细胞因子，IL -1β的刺激下，分别用E -选择素+人血管内皮细胞模型（1-6）。据此前报道，我们观察到强大的KG1a细胞滚动IL -1β刺激的HBMEC - 60细胞，通过一系列在E -选择素依赖性（图 3）（KG1a细胞上滚动时相比差异有统计学意义的剪应力水平非IL -1β刺激HBMEC - 60细胞）。阴性对照，其中包括对KG1a细胞对IL -1β刺激HBMEC - 60细胞或滚动IL -1β刺激HBEMC - 60细胞预处理的抗人E -选择素单克隆抗体，进行并行展示的依赖滚动活动的细胞因子刺激和E -选择素的表达（图3）。此外，滚动分析KG1a细胞预处理与霍乱弧菌神经氨酸酶（唾液酸酶）或与菠萝蛋白酶，非特异性蛋白酶消化表面E -选择素糖蛋白配体，强调E -选择素配体的末端唾液酸残基和表面的糖蛋白的重要性活动（2,3）。

<ol>
	<li>Dimitroff, C. J., Kupper, T. S., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+leukocytic+migration+to+inflamed+skin+with+a+novel+fluorosugar+modifier+of+cutaneous+lymphocyte-associated+antigen.">Prevention of leukocytic migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen.</a> J. Clin. Invest. 112, 1008-1018 .</li><li>Dimitroff, C. J., Lechpammer, M., Long-Woodward, D., Kutok, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin.">Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin.</a> Cancer Res. 64, 5261-5269 (2004).</li><li>Dimitroff, C. J., Descheny, L., Trujillo, N., Kim, R., Nguyen, V., Huang, W., Pienta, K. J., Kutok, J. L., Rubin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+leukocyte+E-selectin+ligands,+P-selectin+glycoprotein+ligand-1+and+E-selectin+ligand-1,+on+human+metastatic+prostate+tumor+cells.">Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells.</a> Cancer Res. 65, 5750-5760 (2005).</li><li>Descheny, L., Gainers, M. E., Walcheck, B., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ameliorating+Skin-Homing+Receptors+on+Malignant+T+cells+with+a+Fluorosugar+Analog+of+N-acetylglucosamine:+P-selectin+Ligand+is+a+More+Sensitive+Target+than+E-selectin+Ligand.">Ameliorating Skin-Homing Receptors on Malignant T cells with a Fluorosugar Analog of N-acetylglucosamine: P-selectin Ligand is a More Sensitive Target than E-selectin Ligand.</a> J Invest Dermatol. 126 (9), 2065-2073 (2006).</li><li>Gainers, M. E., Descheny, L., Barthel, S. B., Liu, L., Wurbel, M., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-homing+receptors+on+effector+leukocytes+are+differentially+sensitive+to+glyco-metabolic+antagonism+in+allergic+contact+dermatitis.">Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis.</a> J. Immunology. 179 (12), 8509-8518 (2007).</li><li>Rood, P. M., Calafat, J., von dem Borne, A. E., Gerritsen, W. R., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+bone+marrow+endothelial+cells:+characterisation+of+new+cell+lines.">Immortalization of human bone marrow endothelial cells: characterisation of new cell lines.</a> Eur J Clin Invest. 30 (7), 618-629 (2000).</li></ol>

平行平板流动腔的分析是一种在体外检测系统专门用于研究白细胞 - 内皮粘合剂相互作用下剪应力条件相似传授那些表面的毛细血管后微静脉。正如在我们的视觉表现描绘，我们展示了这个系统的调查E -选择素的作用的价值 - E -选择素配体介导白细胞圈养和激活微血管内皮细胞的表面上滚动。我们也揭示蛋白酶，糖苷酶和阐明E -选择素及其配体在这个系统中的作用的封闭抗体治疗中的应用。这些分析是高度重现性好，有利于形成学习E -选择素的实验依据 - E -选择素配体在体内的功能。 此外，以研究细胞与细胞因子激活的微血管内皮细胞（HBMEC或人脐静脉或皮肤微血管内皮细胞）的单分子膜的附着力，可以用纯化的重组人E -选择或作为基材的E -选择素，免疫球蛋白嵌合分子检测细胞粘附。胶粘剂等的相互作用，通过白细胞介（L -型）选择和血小板（P）的选择，也可以使用平行板流动腔技术检测。通过改变细胞单层/蛋白底物的类型，或输入的白细胞/选择，转染的细胞模型，研究人员可以研究生理剪应力条件下的这些相互作​​用的作用。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178-500ML</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>0.5 M EDTA</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15575-038</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Parallel-Plate Flow Chamber Kit</td>
<td>&nbsp;</td>
<td>GlycoTech</td>
<td>31-001</td>
<td>&nbsp;</td>
<tr>
<td>PHD2000 Programmable Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>35x10mm Tissue Culture Dish</td>
<td>&nbsp;</td>
<td>BD FALCON</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin , from human plasma</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>86088-83-7 F2006-2MG</td>
<td>&nbsp;</td>
<tr>
<td>Interleukin-1_, Human; Recombinant</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>I9401-5UG</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (Hank s Buffered Saline Solution)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14170</td>
<td>&nbsp;</td>
<tr>
<td>Medium 199 w/ HEPES & Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12320-039</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum</td>
<td>&nbsp;</td>
<td>Innovative Research, Inc.</td>
<td>IPLA-SER1</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Abraxis Pharmaceutical Products</td>
<td>504011</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human fibroblast growth factor</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>233-FB</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Test tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352008</td>
<td>&nbsp;</td>
<tr>
<td>Anti-human E-selectin clone BBIG-E4(5D11)</td>
<td>&nbsp;</td>
<td>R&D Systems	</td>
<td>BBA16</td>
<td>&nbsp;</td>
<tr>
<td>Bromelain</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>B-4882</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
				 	
	

		</div>

analysis of physiologic e-selectin-mediated leukocyte rolling on microvascular endothelium

E -选择是对微血管内皮细胞1型膜蛋白，有助于启动的循环白细胞，皮肤，骨骼和发炎组织的招聘。 E -选择素的表达，是构成对皮肤和骨骼的微血管和促炎性细胞因子，如IL -1α/和TNF -α，微血管发炎组织，诱导。这种外源凝集素受体介导循环白细胞，在一个特点的滚动行为结果与碳水化合物的反受体配体结合的相互作用较弱。因为这些相互作​​用的胶更稳定的事件和diapedesis活动，白细胞滚动活动的特征和白细胞E -选择素配体识别之前已白细胞贩运和炎症的研究和消炎疗法发展的主要目标（1-5） 。本报告的目的是提供一个视觉生理血流条件下E -选择素E -选择素配体相互作用的研究最广泛使用的技术，全面的描述。我们的实验室与哈佛皮肤病研究中心一起，用一个国家的最先进的平行平板流动腔装置伴随着数字可视化和新的录音软件，NIS元素。这项技术使我们能够粘附在屏幕上的可视化，以及记录在视频格式的滚动活动实时事件分析。细胞粘附的参数，如滚动频率，抗剪和有约束力的效率/圈养的，NIS元素的软件计算，导出到Excel电子表格和统计分析。在这里介绍的示范，我们采用的平行板流动腔调查现场人骨髓内皮细胞（HBMEC）E -选择素依赖的白细胞滚动活动。人类造血祖细胞KG1a细胞，表达高水平的E -选择素配体，被用来作为我们的白细胞模式，而一个永生HBMEC细胞株，HBMEC - 60细胞，内皮细胞模型（6）。诱导和模拟原生E -选择素的表达，在流室HBMEC - 60细胞与IL - 1激活。我们的视频演示表明，平行板流分析是一个适合学习生理E -选择素介导的白细胞滚动活动和白​​细胞E -选择素配体（S）的功能特性在流动腔的方法，可确定实施蛋白酶或糖苷酶消解。

Watch this Scientific Journal Video about Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium at JoVE.com

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

这份报告提供了一个平行板流动腔分析研究生理剪切应力下的白细胞内皮细胞相互作用的视像描划。调查的内皮细胞（E） - 选择素和白细胞E -选择素配体触发白细胞在血管内皮细胞表面滚动的作用，这种方法尤其有用。

微血管内皮生理E -选择素介导白细胞滚动分析

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

analysis-physiologic-e-selectin-mediated-leukocyte-rolling-on

Research

JoVE Journal

Biology

19.0K 次观看.  Brigham and Women's Hospital. 这份报告提供了一个平行板流动腔分析研究生理剪切应力下的白细胞内皮细胞相互作用的视像描划。调查的内皮细胞（E） - 选择素和白细胞E -选择素配体触发白细胞在血管内皮细胞表面滚动的作用，这种方法尤其有用。

视频: Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

生物人类免疫缺陷小鼠的微血管网络

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq的凝血酶处理和控制人肺微血管内皮细胞的转录组分析

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.