这项工作得到了德国教育和研究部 (BMBF 01EO1003 和 BMBF 01EO1503) 的支持。

在由莱茵兰-Palatinate (授权号 23 177-07/G12-1-002 和 23 177-07/G15-1-051) 的动物实验伦理委员会批准下, 对8岁至12周龄的雄性小鼠进行了实验。
1. 小鼠麻醉和手术准备
<ol>
	<li>手术前, 检查动物的健康状况。手术前, 每天对动物进行2-3 天的监测, 观察它们的一般情况 (外貌、姿势、自发行为) 以及体重、食物和用水量。</li>
	<li>准备麻醉用咪唑安定 (5 毫克/千克体重), medetomidine (0.5 毫克/千克体重), 和芬太尼 (0.05 毫克/千克体重), 以腹腔 (ip) 注射200µL/30 克鼠标在1毫升注射器与26克针。</li>
	<li>在一个39° c 升温平台的操作场上进行渗透泵植入和动物制剂的综合防治实验。在消毒浴中消毒所有工具, 消毒加温平台。</li>
	<li>在手术过程中, 用眼药膏保护动物眼睛。在渗透泵植入前用剃刀除去毛皮。使用脱毛膏和棉签分离动脉和颈静脉导管植入在综合防治实验。用两种皮肤消毒剂对动物皮肤进行消毒: 一种含有聚维酮碘的防腐剂和消毒剂, 一种含有 octenidine 的皮肤防腐剂。</li>
	<li>准备主混合拮抗麻醉 ("antisedan 混合") 与 atipamezol (0.05 毫克/千克体重) 和马西尼 (0.01 毫克/kgbody 重量), 并准备200µL/鼠标在1毫升注射器与 26 G 针是皮下注射 (南卡罗来纳州) 注射。</li>
</ol>
2. 渗透泵的制备和植入
注: 使用渗透泵的皮下 ii 输液详细介绍了鲁et al.17
<ol>
	<li>用无菌盐水重组冻干粉制备 ii, 并以动物重量和泵送率调节浓度。将重组的 ii 在塑料管中 (不要使用玻璃管)。</li>
	<li>用 ii 溶液填充泵, 以提供1毫克/(kg·d) 7 天。准备好后, 将泵在无菌盐水中保持在37° c 的4-6 小时以上。</li>
	<li>在麻醉后用后足反射法对小鼠进行完全麻醉, 并将其置于温热的操作场。麻醉诱导需要10-15 分钟, 如果把动物放在黑暗环境中, 效果会更好。</li>
	<li>剃掉老鼠的下背部, 用酒精性皮肤防腐剂消毒。</li>
	<li>用剪刀将1厘米的切口垂直于脊柱。使用海峡止血为泵创建一个口袋, 并插入泵 (流版主第一)。口袋必须允许泵的自由运动, 在伤口上没有压力。</li>
	<li>用缝线缝合伤口, 而不是夹紧, 以限制炎症;然后用皮肤防腐剂消毒。</li>
	<li>拮抗麻醉的200µL 的 antisedan 混合和返回的老鼠到它的笼子。</li>
	<li>用丁丙诺啡 (0.075 毫克/千克) 进行术后镇痛, 一次手术后根据动物的行为要求。</li>
</ol>
3. 颈静脉导管植入及动脉制剂
<ol>
	<li>对麻醉后的小鼠进行全身麻醉, 用后部食物反应, 在39° c 手术板上用直肠探针将麻醉动物放在背 recumbence 上, 以保持体温并把药膏放在在程序中保护他们的眼睛。</li>
	<li>使用脱毛膏去除颈部毛发。用棉签将奶油涂抹2分钟, 直到头发开始脱落。再加2分钟后, 用刮刀除去奶油和毛发, 用酒精性皮肤杀菌消毒皮肤。</li>
	<li>在显微镜下进行手术。做一个 1-1. 5 厘米长的切口在皮肤纵向地在脖子, 1 cm 在气管旁边。然后使两个其他切口垂直于第一切口的两个肢。</li>
	<li>小心地移除皮肤旁边的组织, 取出覆盖左颈静脉和气管的皮肤块。</li>
	<li>仔细隔离腮腺;舌下和颌下腺腺体从感兴趣区与2弯曲钳。不要割伤或损伤腺体, 让它们进入颈静脉和动脉的最佳通路。</li>
	<li>为了隔离颈静脉, 使用薄钳, 并慢慢打开和关闭它, 轻轻地释放血管从周围的组织。一旦静脉是完全清洁, 定位钳下的船只, 并放置两个7-0 缝合10厘米每下。</li>
	<li>用两节结缝合头部近端。在其他缝合, 准备一个结, 但不要关闭它。</li>
	<li>保持导管 (0.28 毫米内径, 0.61 毫米外径), 用一只手钳填充37° c 无菌生理盐水;而另一方面, 用一把薄薄的剪刀或一条弯曲的26克针, 在容器中做一个小切口。然后将导管插入颈静脉, 并闭合两次结节。在导管上缝合头部近端, 以确保完全固定。</li>
	<li>为了分离和准备颈动脉, 用薄弯曲钳解剖覆盖气管的区域。</li>
	<li>一旦动脉从周围的组织中解脱出来, 从血管中取出迷走神经 (它看起来像一条与颈动脉相邻的白线) (但不要割断它)。薄钳可以关闭, 慢慢打开, 轻轻地调动神经。</li>
	<li>一旦两个动脉都完全清洁, 放置钳下第一颈动脉, 并放置一个小的黑色3毫米宽 x 2.5 厘米长的塑料件下的容器。这是非常重要的, 不过度的船只和检查, 血流存在, 一旦船舶持有人被放置。将第二个动脉放在容器的支架上。如果未将鼠标直接置于显微镜下, 则将腺体放回气管上, 并应用37° c 无菌生理盐水以避免干燥区。</li>
</ol>
4. LysM 和粘附细胞的评价
<ol>
	<li>将吖啶橙从2毫克/毫升的库存溶液中贮存在-20 ° c, 并在1毫升注射器中稀释1:4 的无菌盐水 (0.5 毫克/毫升最终浓度)。</li></ol>保护注射器免受光照, 每只老鼠使用200µL。<ol>
		<li>测试不同条件: (1) 用颈静脉导管注射吖啶橙;(2) 将吖啶橙直接注入尾侧静脉 (无颈静脉导管植入);(3) 无吖啶橙的测量使用荧光 LysMCre+表意文字+小鼠 (这里再次没有颈静脉导管植入)。</li>
	</ol>
	
	<li>一旦导管到位, 两个动脉被隔离, 将鼠标置于显微镜下。使用远距离冷凝器和 10X (NA 0.3) 水浸泡物镜, 用单色仪、分束器和电荷耦合器件摄像机对高速宽场荧光显微镜进行测量。利用 real-time 成像系统进行图像采集和分析。</li>
	<li>将显微镜物镜集中在血管内皮表面的颈动脉中央。缓慢注入50µL 吖啶橙 (0.5 毫克/毫升) (考虑到第一次注射的导管死体积)。将软件设置为记录100图像, 每个图像的曝光时间为120毫秒。</li>
	<li>每一个视频, 每颈动脉 (左, 右) 在不同的位置制作四视频。如果荧光信号减弱, 则再注入50µL 吖啶橙。</li>
	<li>录制完所有的视频后, 安乐的动物被颈椎脱位。</li>
	<li>将视频速度设置为每秒10图像, 并在每个视频的容器中间放置一个200µm x 250 µm 矩形视图中的滚动和粘连单元格的数量。计数轧制和黏附细胞;黏附细胞被定义为在十年代视频中不移动或脱离内皮的细胞。为了简化量化, 用红色荧光去除通道, 只使用绿色荧光计数轧制和粘附细胞。</li>
	<li>为实验使用吖啶橙, 做一个手动阈值的荧光, 以消除背景, 由内皮细胞。</li>

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作者没有透露

LysM+单核细胞曾被证明与高血压的发展有牵连5。在这里, 我们表明, LysM 的免疫细胞的+ , 并坚持到内皮层, 以响应 ii 输液。此发现是使用 LysMCre+表意文字+小鼠获得的, 从我们以前的研究中探索免疫细胞在高血压中的作用在体内通过对吖啶橙注射液的可视化白细胞,6,10。因此, 细胞类型的黏附在内皮的歧视是不可能的。
这是一个非常有用的工具的血管研究和体内观察细胞直接在血管, 但注射染料的影响, 在一个外科插入导管的帮助下, 高血压炎症的背景仍然未知。为了评价导管和吖啶橙注射液的潜在作用, 我们利用了 LysMCre+表意文字+鼠标。ii 输液增加了滚动的 LysM+细胞的数量到内皮。插入导管进入颈静脉放大的效果和更多的滚动和粘附白细胞检测 ii 注射小鼠导管与无导管植入的小鼠相比。这表明, 植入颈动脉导管导致全身炎症反应的伤口愈合的情况下覆盖与免疫反应看到的高血压18。此外, 由于颈静脉靠近颈部动脉, 另外的免疫活化可能是通过影响颈静脉而发生的。由于这种作用是不存在的, 当注射直接进入尾部静脉, 我们可以假设, 该效应不是由于染料, 而是插入导管的程序。我们在这里展示了转基因的报告鼠的重要性, 表达荧光蛋白, 以不干涉在体内过程中的实验。此外, 尾部静脉注射荧光示踪剂可能是一个可能的替代静脉导管注射。
该过程的一个关键步骤是 ii 输液。尾袖评估的血压可以作出, 以控制的有效性, ii 交付的泵。2−3天后血压应增加。为了限制可能影响 LysM+细胞活化的炎症, 在植入泵的切口处, 应该用缝合线而不是夹子来做。必须注意的一个限制是尾静脉注射: 为了使最好的数据获取, 4 视频通常是为每一个颈动脉。如果荧光信号下降, 50 µL 吖啶橙注射, 但与尾部注射更难进行多次注射和保持动脉稳定在显微镜下的目标。颈静脉导管存在的持续时间也可能调节免疫细胞的活化作用, 因为它影响了在导管植入后4小时内制备的富含血小板血浆的凝血活化19。这方面的导管植入需要进一步的调查。
最后, 即使我们赞赏颈静脉导管植入对 LysM 的影响, ii 输液后, 我们不能完全估计的准备, 皮肤移除和颈动脉隔离在潜在的 LysM +细胞的作用激活.我们的结论是, 使用荧光报告鼠像 LysMCre 的+表意文字+鼠标建议, 以避免破坏船只的完整性在动物准备为在体内成像。

动脉高血压增加了心血管疾病和死亡的风险, 促进动脉粥样硬化, 冠心病, 动脉或静脉 thromboembolisms 的发展1。高血压的发展取决于环境、遗传、内分泌和血流动力学因素的相互作用。目前, 免疫及相关炎症在高血压病因中起重要作用2。
在免疫细胞, T 淋巴细胞以及单核和巨噬细胞被发现是因果关系的 ii 诱导的血管炎症和高血压, 部分与他们的能力触发活性氧的物种3。巨噬细胞集落刺激因子缺乏小鼠表现出减少对 ii 的反应血压升高和血管炎症4。在以前的工作中, 我们可以证明 LysM + 单核细胞驱动血管功能障碍和炎症的 ii 诱发高血压5。最近, 我们描述了一个新的途径, 凝血因子 XI 配合血小板和血管壁诱导凝血酶依赖性血管炎症6。目前有关免疫系统在高血压中的作用的知识最近总结和回顾了罗德里格斯-Iturbe et al。7
由于免疫细胞参与高血压的发展变得明显, 研究血管与免疫细胞相互作用的模型和技术成为必要。荧光的血管综合病是一个有用的工具, 观察体内循环血细胞和内皮之间的相互作用,8,9,10。用这种技术, 注射染料插与脱氧核糖核酸 (例如吖啶橙色) 能形象化有核的细胞 (循环和从内皮)。在动脉或静脉损伤模型中, 可以注入 rhodamin-6G 或ˊ (现金流量) 的分离血小板, 以显示血小板丰富的血栓. 
通常, 颈静脉导管用于注射示踪剂或标记血小板。改变内皮和随后活化的凝血叶栅都知道有影响单核细胞活化。内皮损伤立即导致血小板活化通过内基质分子密封的组织, 随之而来的单核细胞吸引和激活11。相反, 一个完整的内皮细胞是已知的抗凝血特性 (例如,通过组织因子途径抑制剂或血栓调节蛋白)12和直接抑制作用的单核,例如, 通过分泌胞外囊泡含有 rna13。单核细胞是已知的产生组织因子, 外源性的凝血级联和表达蛋白酶活化受体 (部), 可以激活凝血酶和参与单核激活14,15.因此, 任何血小板活化或由于血管损伤引起的凝血级联, 可能对单核细胞活化产生意想不到的影响, 并干扰观察到的现象。在表意文字转基因 LysM 转基因小鼠的帮助下, 一个 double-fluorescent 的报告鼠 (LysMCre+表意文字+), 我们建议详细研究注射用导管的效果和替代方法在 LysM+粒细胞在小鼠动脉高血压模型中的作用16。

<table>
	<tbody>
		<tr>
			<td>Midazolam</td>
			<td>Ratiopharm GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Pfizer Deutschland GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Janssen-Cilag GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Alzane</td>
			<td>Zoetis</td>
			<td></td>
			<td>Antisedan mix</td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Hikma pharma</td>
			<td></td>
			<td>Antisedan mix</td>
		</tr>
		<tr>
			<td>Braunol</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Octeniderm</td>
			<td>Sch&#252;lke</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic pumps</td>
			<td>Alzet</td>
			<td>1007D</td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>Angiotensin II</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>7-0 prolene suture</td>
			<td>Ethicon</td>
			<td></td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>Acridine orange</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Smiths Medical Deutschland GmbH</td>
			<td></td>
			<td>Inside diameter, 0.28 mm; outer diameter, 0.61 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI fluorescence microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam Splitter</td>
			<td>Photometrics</td>
			<td>CMR-DV2-SYS - DualView2 MicroImager</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UMPLFLN10X</td>
			<td>Water immersion objective with a monochromator</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>ORCA-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and analysis software</td>
			<td>Olympus</td>
			<td>Realtime Imaging System eXcellence RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>Jax 008705</td>
			<td>B6.Cg-Tg(CAG-DsRed,-EGFP)5Gae/J</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>Jax 004781</td>
			<td>LysM Cre (B6.129P2-Lyz2tm1(cre)Ifo/J )</td>
		</tr>
	</tbody>
</table>

visualizing leukocyte rolling and adhesion in angiotensin ii-infused mice: techniques and pitfalls

荧光活体视频显微镜 (综合防治) 的血管是一个既定的方法来评估免疫细胞的活化作用和他们的能力, 并坚持内皮层。通常使用荧光染料或荧光偶联抗体的注入来可视化循环细胞。另外, 还可以使用荧光报告小鼠。白细胞的相互作用, 特别是溶菌酶 M+ (LysM+) 单核分子, 血管壁在促进血管功能障碍和动脉高血压方面起着举足轻重的作用。我们在这里提出的技术, 以可视化和量化白细胞轧制和粘附在血管紧张素 II (ii) 诱导高血压的小鼠通过综合防治。
导管的植入会损害血管壁, 导致改变的血细胞反应。我们比较了不同的注射技术和管理路线, 以形象化的白细胞在一个 LysMCre 的+表意文字+小鼠广泛表达红色荧光蛋白和绿色荧光蛋白的条件表达在 LysM+单元格。为了研究 LysM 的+细胞活化, 我们使用了 ii 灌注小鼠, 其中白细胞的滚动和粘附增加到内皮。我们要么通过静脉导管注射吖啶橙, 要么直接通过尾静脉, 比较滚动和黏附细胞的数量。我们发现, 颈静脉导管植入本身增加了在假灌输的 LysMCre+表意文字+小鼠与对照组相比滚动和黏附 LysM+细胞的数量。这种活化作用增强了 ii 的小鼠。有趣的是, 直接通过尾静脉注射吖啶橙并没有增加 LysM 的+细胞粘附或在假灌注小鼠中的滚动。因此, 我们证明了转基因小鼠表达荧光蛋白在实验过程中不干扰体内过程的重要性。此外, 尾部静脉注射荧光示踪剂可能是一个可能的替代静脉导管注射。

动脉的 LysMCre+表意文字的+小鼠注入 ii 的观察使用了综合病媒。用颈静脉导管注射吖啶橙。我们的目的是观察与血管壁接触的 LysM+细胞与所有有核循环细胞的比例 (这也可能与血管相互作用, 因为细胞类型与血管相互作用的区别仍然是一个开放我们以前的工作的问题)。在基线情况下, 颈静脉导管的出现会导致 LysM 的+细胞 (图 2A, D) 的粘连。吖啶橙注射液后, 相同的细胞是荧光的, 而且内皮细胞是荧光的 (图 2B, E)。在减少内皮相关荧光的背景下, 数据表明所有有核的黏附细胞是 LysM 的+ (图 2C, F);这些结果在 ii 输液确认我们以前的结果并证明 LysMCre 的+表意文字+是观察 ii 对 LysM+单元激活的影响的好模型后才成立。
在 LysMCre+表意文字+中, 我们评估了吖啶橙在 ii 输液后的 LysM+细胞活化中的作用。在一周的 ii 输液后, LysMCre+表意文字、+小鼠颈动脉内的白细胞内皮相互作用通过经颈静脉导管或通过尾静脉的吖啶橙注射液进行影像和可视化。没有任何导管或注射, LysM 的滚动的+细胞是显着增加后, ii 输液相比, 未经处理的小鼠, 和粘连显示增加 (图 3)。注射吖啶橙与颈静脉导管增加了 ii 治疗小鼠的黏附力和滚动, 更大程度上与没有导管的小鼠相比 (图 3)。与未接受吖啶橙注射液的小鼠相比, 尾静脉注射吖啶橙会导致类似的粘连和轧制 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56948/56948fig1.jpg"> 
图1。血管紧张素 II. 的输液方案及对 LysM 的评价+细胞在小鼠体内滚动和黏附。LysMCre+表意文字+小鼠注入了7天, ii 和黏附, 以及滚动 LysM+细胞在动脉是量化的, 或没有注射吖啶橙通过颈静脉导管或尾静脉注射.<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56948/56948fig2.jpg"> 
图2。评价 LysM 的+细胞黏附和滚动在颈动脉 LysMCre+表意文字+鼠标。粘附 LysM+ LysMCre 中的细胞 + 表意文字 + 鼠标前 (a, D) 和后 ( B, E) 吖啶注射液通过颈静脉导管。红色和绿色荧光被记录, LysM 的+细胞 (绿色) 和平滑肌细胞 (红色) 是可见的。注射吖啶橙后, 内皮细胞也可在绿色可见, 但去除背景荧光只允许循环有核细胞可视化 (C, F)。<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56948/56948fig3.jpg"> 
图3。血管紧张素 II. LysMCre 中荧光染料的管理路线评价+表意文字+小鼠。滚动的量化 (A) 和黏附 (B) LysM+细胞在假操作或 ii 注入动物, 并有或不注射吖啶橙使用颈静脉导管或尾静脉注射。不同条件 (C) 的代表性图片。结果均值平均±标准误差。执行了2路方差分析和 Bonferroni 的post测试, n = 3-12/组;*p和 #60; 0.05。<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls at JoVE.com

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

这份手稿描述了使用转基因的记者小鼠和不同的管理途径荧光染料在血管紧张素 II 诱导高血压使用活体视频显微镜的血管评价免疫细胞的活化作用及其能够滚动和坚持内皮。

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

visualizing-leukocyte-rolling-adhesion-angiotensin-ii-infused-mice

Research

JoVE Journal

Immunology and Infection

8.6K 次观看.  University Medical Center Mainz. 这份手稿描述了使用转基因的记者小鼠和不同的管理途径荧光染料在血管紧张素 II 诱导高血压使用活体视频显微镜的血管评价免疫细胞的活化作用及其能够滚动和坚持内皮。

视频: Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

甲病毒转导系统:可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

成像白细胞粘附到血管内皮细胞在高腔内压力

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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

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

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

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

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...