这项工作得到了瑞士国家基金会 (SNF) 赠款 (176124、145038和 148183)、欧洲委员会居里夫人重返社会补助金 (612742) 和 SystemsX.ch D.U.P. (2013/124) 赠款的支持。

所有涉及老鼠的动物程序都是按照瑞士联邦兽医局的指导方针和动物协议进行的, 当地兽医当局已经批准。
1. CD11c-YFP 小鼠的流感感染
<ol><li>安全性 注: Rico/8/34 甲型h1n1 流感 (PR8) 的老鼠适应菌株生长在受精卵, 纯化和滴定如前所述21。根据生物安全水平 (BSL) 2 条件, 在生物安全柜下进行了涉及受感染动物或生物样品的所有步骤。<ol>
<li>在感染程序前后用70% 乙醇溶液清洁生物安全柜。</li>
		<li>根据世卫组织生物安全准则 (http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf), 丢弃在此过程中产生的所有废料。在装满70% 乙醇溶液或医用消毒剂的塑料袋中, 丢弃耐高压箱和污染液体中的固体废弃物。</li>
	</ol></li>
	<li>流感鼻腔感染 注: B6。本研究采用 C57BL/6J 背景的 Cg Tg (Itgax-金星) 1 mnz/J (CD11c-YFP)22 。在生物医学研究研究所, 小鼠被保存在特定的无病原体设施中。<ol>
<li>每笼最多可放置5岁及性别配对 (六至八周) CD11c-YFP 鼠。在感染程序之前, 至少要等待两天的时间, 让老鼠适应住房条件。</li>
		<li>解冻 PR8 的库存, 并准备相应的稀释使用冷 1x Dulbecco 的磷酸盐缓冲盐水改性不含氯化钙和氯化镁 (PBS), 以获得最终浓度的200斑块成型单位 (PFU) 在30µL. 保持病毒稀释在整个过程中的冰。 注: 建议在使用前对每批流感病毒进行滴定, 以确保准确的感染剂量。</li>
		<li>使用26克针1毫升注射器注射剂量氯胺酮 (100 毫克/千克) 和甲苯噻嗪 (10 毫克/千克) 腹腔 (ip)。</li>
		<li>等待, 直到鼠标完全麻醉 (完全丧失矫正和踏板撤退反射)。深层麻醉对最佳感染是必要的, 因为没有完全麻醉的小鼠会吞下或排出病毒接种, 导致感染剂量的变化。</li>
		<li>将麻醉鼠置于仰卧位。收集15µL 病毒接种。把吸管尖端靠近鼠标左鼻孔, 并免除病毒接种下降。因为必须吸入水滴, 不要直接在鼻腔内吸管。</li>
		<li>等待2–5分钟, 并放置在右鼻孔中含有其余15µL 病毒接种的吸管尖端。 注意: 为了避免窒息, 在大约二十年代的时间间隔内分配小尺寸滴。</li>
		<li>检查鼠标是否正确呼吸并将其放置在侧压卧笼中。监测小鼠呼吸和麻醉恢复 (诱导后约60分钟)。 注意: 同时感染1只老鼠是可能的。在这种情况下, 管理病毒接种在所有老鼠的左鼻孔序列, 等待2–5分钟, 然后管理病毒在右鼻孔。为了避免个体间的变异, 在同一时间感染不超过3只老鼠。</li>
		<li>每天监测动物健康状况和体重损失。</li>
		<li>弄死小鼠根据权威指南确定的人道端点。安乐死方法必须得到动物实验当局的批准, 并要尊重当地的道德规范。经过双光子实验, 弄死小鼠用药过量的氯胺酮/甲苯噻嗪后颈椎脱位。在所有其他实验中, 使用 CO2吸入作为安乐死的方法。 注: 为了测量受感染气管的病毒滴度, 50% 种组织培养感染剂量 (TCID50) 检测或实时聚合酶链反应 (rt-pcr) 检测可进行如前所述23。</li>
	</ol></li>
	<li>流式细胞术评价中性粒细胞在气管内的应用 注意: 协议的这一部分是可选的。目的评价在流感感染后气管黏膜中中性粒细胞的招募情况。<ol>
<li>弄死感染和未感染的控制小鼠在3天后感染 (私家侦探) 通过 CO2吸入。 注意: 为了防止气管损伤, 避免使用颈椎脱位弄死动物。此外, 预防安乐死小鼠的灌注是可取的, 以避免血液中性粒细胞的污染。</li>
		<li>用70% 乙醇溶液喷洒鼠标颈部, 用手术剪刀从胸部到下巴进行皮肤切口。</li>
		<li>用镊子将涎腺分开, 并暴露气管。</li>
		<li>用镊子和剪刀解剖气管周围的肌肉。 注意: 这一步必须小心执行, 因为气管在过程中很容易损坏。</li>
		<li>用钳夹住气管, 并通过解剖仔细分离食道。</li>
		<li>用镊子将气管的胸腔部分托住, 在支气管树的开始处进行切口。然后把气管从喉部分离出来, 然后小心地取出剩下的肌肉。</li>
		<li>将该器官放置在1.5 毫升管中, 其中含有 RPMI 1640 + HEPES 介质 (RPMI) 在冰上。</li>
		<li>制备用于气管消化的酶合剂, 在 RPMI 中含有0.26 单位/毫升胶原酶 (i 和 II) 和0.2 毫克/毫升的 DNase。</li>
		<li>将解剖气管放置在含有1毫升酶合剂的6井板中。</li>
		<li>在镊子和剪刀的帮助下, 把风琴切成小块。在这一步, 把盘子放在冰上。</li>
		<li>孵育37°c 45 分钟。在这段时间里, 每隔15分钟摇动盘子。</li>
		<li>在 PBS 中加入1毫升的 (2 毫米 EDTA 和2% 种热灭活过滤-灭菌胎牛血清), 停止酶消化。</li>
		<li>用1毫升的吸管并用重悬溶液, 帮助解除部分未消化的组织。</li>
		<li>通过40µm 过滤器传递内容, 将解决方案转移到另一个井中。然后, 用2毫升注射器活塞轻轻粉碎被困在滤网顶部的器官的其余部分。 注: 在过滤器上粉碎部分未消化的气管是在流动细胞分析中获得最佳细胞数的关键步骤。</li>
		<li>清洗井和过滤器与外地资产管制系统洗涤缓冲和转移悬浮在5毫升管保持在冰上。</li>
		<li>离心管在 166 x g 5 分钟在4摄氏度。丢弃上清液, 并用重悬100µL 的细胞。</li>
		<li>对流式细胞术进行抗体表面染色。简单地, 使用抗体对 CD16/32 的隔离细胞阻断 Fc 受体, 其次是表面染色。为了正确识别中性粒细胞, 流式细胞术小组应包含以下抗体: αLy6G, αCD11b, αCD45, 以及一种能排除死细胞的活性染料。</li>
		<li>在流式细胞仪上运行样本的全部内容并分析数据。<ol>
<li>为了获得最佳的免疫细胞数, 以不超过3000个事件的速度运行单细胞悬浮。这将减少在购置过程中排除的事件数。使用这个协议, 应该可以获得每气管1到200万的细胞。</li>
		</ol>
</li>
	</ol></li>
</ol>2. 中性粒细胞的分离和注射
注意: 在这个过程中, B6.129 (ECFP) 的 CK6Nagy 和 J 小鼠 (CK6-ECFP) 使用了24。这些动物表达 CFP 在所有细胞类型下的人β肌动蛋白启动子。另外, 也可以使用 C57BL/6J 小鼠隔离细胞并根据步骤2.6 中描述的协议对其进行染色。中性粒细胞的纯化和操作可能会增加其活化状态, 有可能改变它们的迁移和功能特性。
<ol><li>弄死动物由 CO2吸入。</li>
	<li>取出股骨和胫骨, 用镊子轻轻擦拭。</li>
	<li>切骨骨骺和冲洗骨髓使用1毫升注射器填充无菌冷 PBS, 在50毫升的管保持在冰上。</li>
	<li>用18克针并用重悬细胞, 用40微米滤网过滤细胞悬浮液。以 110 x g为5分钟, 在2毫升的冷 PBS 中, 用 PBS 在4摄氏度和并用重悬细胞上洗涤一次。</li>
	<li>
稀释 100% Percoll 9:1 在 10x pbs 和准备梯度解决方案的 Percoll 72%, 64% 和52% 使用 1x PBS。小心层2毫升的每一个三梯度在15毫升管, 从最集中的一个开始。
	<ol>
<li>仔细添加2毫升的骨髓细胞悬浮在梯度和离心机在 1100 x g 30 分钟室温 (RT) 没有加速度和刹车。</li>
	</ol>
</li>
	<li>小心地删除64% 和72% 之间的接口, 并在 200 x g清洗一次在4°c 与冷 PBS。并用重悬细胞在 100 ul 的 PBS, 并保持在冰上。中性粒细胞的纯度预计将高于90%。</li>
	<li>或者, 使用制造商的协议, 用细胞增殖套件标记中性粒细胞。将染料添加到细胞悬浮液中, 最终浓度为10微米, 体积为1毫升, 孵育37°c 30 分钟, 洗涤 (166 x g, 5 分钟)。</li>
	<li>用 hemocytometer 和静脉注射 5 x 106细胞, 在先前受感染的 CD11c-YFP+小鼠中使用26克针1毫升注射器, 在成像前, 用一根最大体积的 100 ul 来估计细胞浓度。</li>
</ol>3. 准备鼠标成像
<ol><li>
麻醉
	<ol>
<li>麻醉感染 CD11c-YFP+鼠3天的私家侦探根据步骤1.2.3。</li>
		<li>一旦老鼠被深深麻醉, 准备一导管麻醉再灌注。<ol>
<li>用镊子从注射器中取出30克针。</li>
			<li>将针插入20厘米 PE-10 医用硅管。</li>
			<li>在管的另一侧插入30克氯胺酮/甲苯噻嗪混合物填充的针头注射器。</li>
			<li>卸下管子内的空气。</li>
		</ol>
</li>
		<li>准备一个20克导管连接到一个管道从氧 insufflating 机, 以保持自动通风 (图 1Ai)。将其设置为130节拍每分钟 (b.p.m.) 的呼吸比率, 其潮汐体积为0.2 毫升, 使用100% 氧气供气。</li>
	</ol>
</li>
	<li>
准备手术用鼠标
	<ol>
<li>将鼠标放在特定的定制手术板上 (图 1Aii iii) 在加热板上, 或在手术期间设置在37摄氏度的加热手术台上。</li>
		<li>使用电动剃刀和脱毛霜 (图 1Bi) 剃去老鼠颈部的毛发。
</li>
		<li>将鼠标放在塑料鼠标位置的上方 (图 1Aii a), 使动物头部处于定位外 (图 1Bii), 以创建一个有利于气管插管的角度。</li>
		<li>用手术胶带固定前肢、爪子和尾巴, 用维生素 a 浓缩凝胶滋润老鼠眼睛, 并对鼠标颈部进行消毒 (图 1Bii)。</li>
	</ol>
</li>
	<li>
手术
	<ol>
<li>准备适当的蒸压物品, 即剪刀、显微外科剪刀和镊子。</li>
		<li>在颈部的长内侧轴上做一个小切口, 大约1厘米长, 在上胸部和穿过下颌下角的线之间的中间区域 (图 1Biii)。</li>
		<li>侧移皮肤斑块和涎腺, 并可视化气管, 由肌肉覆盖。然后, 用镊子仔细解剖气管肌肉 (图 1Biii)。</li>
		<li>用导管插管鼠标, 开始人工通气 (图 1Biv)。 注: 导管由外部塑料部分组成, 用于保护气管粘膜和内铁针。针的存在是延长和稳定气管和调节其博览会的理想解决方案。人工通气通过导管将保证正确的呼吸鼠标。</li>
		<li>立即启动人工通风监测鼠标呼吸。</li>
		<li>用手术钩 (图 1Bv) 连接到手术板上的特定杆上, 固定导管高度和方向 (图1Aii b)。将气管暴露在下巴的同一高度。</li>
		<li>用石油果冻将气管围起来, 用几滴预加热的 PBS 盖住, 保证适当的水合器官 (图 1Bvi)。</li>
		<li>在准备工作的顶端安装盖玻片。在这个步骤中, 将盖玻片粘附在金属支架上 (图 1Bvii), 这将被拧到 XYZ 翻译 (图 1A二 c)。调整 XYZ 翻译器, 将盖玻片放在手术准备的顶端。</li>
		<li>放置导管用于麻醉腹腔的管理 (图 1Bviii)。</li>
		<li>注射氯胺酮/甲苯噻嗪混合物的初始剂量的50% 每30分钟。 注: 从50% 至25% 的初始氯胺酮/甲苯噻嗪混合物中再加药是一种安全有效的替代品25。然而, 由于该协议是终端和严格的固定化的动物是需要在整个过程中, 使用更高的剂量, 以维持一个深的手术平面的麻醉。</li>
	</ol>
</li>
</ol>4.体内时间推移成像
注: 图像采集采用直立双光子显微镜, 配备两个 Ti: Sa 激光器, 温度控制孵化室, 25 x/NA 1.1 水浸泡目标。用于图像采集的光电倍增管 (PMT) 要么是混合探测器, 要么是高灵敏度 GaAsP。
<ol><li>将手术板与麻醉鼠标放在显微镜孵化室内 (在37–38°c 预热), 并在盖玻片上加一滴水。</li>
	<li>中心, 并发现重点在气管组织。</li>
	<li>
将扫描频率设置为800赫兹, 具有 520 x 520 像素, 视场为 440 x 440 微米2 , 直线平均值1。
	<ol>
<li>调谐 Ti: Sa 激光到 830 nm 激发第二次谐波产生 (SHG) 从胶原蛋白, 与指示力在源150兆瓦。调整第二 Ti: Sa 激光在920毫微米与94兆瓦在来源, 刺激 CFP 和 YFP。</li>
		<li>同时励磁3D 和 timelapse 采集模式的建立 注意: 保持激光功率尽可能低, 以最小化漂白和毒性。</li>
	</ol>
</li>
	<li>记录荧光使用两个通道在非 descanned 模式, 与主分色镜在 560 nm。在本协议中使用的设置中, 第二个分色镜将信号分割为 495 nm, 将通道 1 (发射过滤器 475/50) 与通道 2 (发射过滤器 525/50) 分开 (图 2A)。该配置从第一通道中的胶原蛋白收集 SHG 光, 而在通道1和2中收集 CFP 发射, 而 YFP 仅在2通道中收集。</li>
	<li>沿 Z 轴定义一个范围50微米, 其步长为3微米 (体素尺寸0.86 微米 x 3 微米)。每年三十年代记录图像的总持续时间为30分钟。</li>
	<li>如果需要, 则获取多个区域。在运行更多的收购, 检查生命体征和注入麻醉通过导管, 如果需要 (见步骤 3.3.10)。</li>
	<li>在成像过程结束时, 弄死小鼠通过氯胺酮/甲苯噻嗪过量后颈椎脱位。</li>
</ol>5. 中性粒细胞-DC 运动和相互作用的图像处理和定量分析
注: 在本协议中, 使用专门的成像软件对显微数据进行分析。
<ol><li>完成4D 图像采集后, 将文件 (数据和元数据) 传输到具有足够计算资源的工作站 (建议的最低系统要求:32 GB RAM、快速固态驱动器、最近的 CPU、专用 GPU 基于大规模并行体系结构)。</li>
	<li>打开图像软件中的文件。</li>
	<li>播放视频并确保感兴趣的单元格清晰可见, 并且不存在图像工件。 注: 为此, 验证样品的运动和亮度变化是否有足够的限制。事实上, 这些都代表了自动分析26的挑战。如果相邻帧之间的样本移动过大, 则应用漂移校正方法, 例如, 将 SHG 通道用作固定参照。这样可以更好地测量细胞的运动, 而不是样本的运动。此外, 在明亮的背景或碎片的存在, 修剪重建的表面使用体积作为选择参数。</li>
	<li>为 CFP+细胞生成一个联合定位通道, 以便在 CFP 和胶原蛋白 (SHG) 之间分离信号。为此, 表示一个浇口多边形 (图 2Bi), 它只选择在绿色通道和蓝色通道中具有正强度的素。 注意: 这个过程可能会因显微镜的过滤器集和细胞的染色而异。它可以通过选择只有在绿色和蓝色通道中具有足够强度的素来实现。在可用的工具中, "coloc" 功能可用于自动计算基于强度阈值的定位信道。此外, 基于机器学习的方法可用于这一步骤与专家27的监督。</li>
	<li>检测和跟踪 CFP+单元格, 使用自动曲面重建和跟踪 (表面工具) 在适当的共定位通道。 注意: 可能需要手动精选最终跟踪错误和排除跟踪时间短于定义的阈值 (即150s)。</li>
	<li>为 CD11c-YFP+单元格生成一个联合定位通道, 以便分离 CFP 和 YFP 信号。为此, 表示一个浇口多边形 (图 2Bii), 它只选择在绿色通道中具有正强度的素, 而蓝色通道中的强度较低。 注: 代表性显微照片显示1通道、通道2、CFP 的共用定位通道、YFP 的共用定位通道以及所有通道的组合, 可在图 2Biii中找到。</li>
	<li>重建 CD11c-YFP+单元格的表面, 并跟踪它们的位置 (曲面工具)。 注意: 此步骤不需要手动更正最终跟踪错误。事实上, 由于 DC 的复杂时空动力学, 从 2 p-病媒数据中重建其精确表面是一项具有挑战性的任务, 可用的分割软件无法实现。在使这项任务具有挑战性的原因中, 薄突起和亮度变化不允许使用某些图像处理技术, 如平滑滤镜或静态阈值。此外, 在 DC 的网络中, 很难将单个细胞仅基于其在 2 p 病媒数据中的出现而分离。由于这些原因, 我们建议重建直流非精确曲面, 而不是通过调整软件参数来进行精确的分割。然后, 通过稳健的指标处理可能的错误, 如5.8 所述。</li>
	<li>
测量单元格迁移。
	<ol>
<li>导出 CFP+和 CD11c-YFP+单元格的经典迁移措施。其中, "轨道速度" 表示细胞的平均迁徙速度, 而 "轨道直线度" 则表示细胞的方向性。这些度量值可以从映像软件导出为电子表格文件。</li>
		<li>在导出的电子表格文件中, 计算 CFP+和 CD11c-YFP+单元格的 "校正轨道直线度" (也称为修正约束比率), 定义为 "轨道直线度" 乘以平方根"跟踪持续时间" 除以视频持续时间的平方根。这一措施比 "轨道直线性" 在短轨道28的存在更健壮, 起源于例如从跟踪错误。 注意: 只有相同长度的视频可以与此度量值进行比较。</li>
	</ol>
</li>
	<li>
测量细胞的相互作用。
	<ol>
<li>如果 CFP+单元格和 CD11c-YFP+单元格之间的距离 (最近的素) 小于或等于阈值 (即2 微米), 则定义一个联系人。 注意: 此阈值应足够严格, 以便仅在单元格接近时检测到联系人。然而, 我们鼓励保持这个阈值大于 0, 理想的 n 乘以体素半径 (n > 2), 因为边界平滑可以使细胞重建小于实际的细胞大小。</li>
		<li>计算 CFP+和 CD11c-YFP+单元格之间的联系人的数量和持续时间。在成像软件中, 这可以通过执行 "接吻和运行" 插件来完成。这些度量值可以从 "统计" 选项卡导出为电子表格文件。</li>
	</ol>
</li>
	<li>在统计软件中导入以前计算过的度量值, 生成绘图并执行统计测试。</li>
</ol>

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作者没有什么可透露的。

这项工作提供了一个详细的协议, 以生成4D 图像显示过继转输转移中性粒细胞的迁移及其与 DC 的相互作用, 在流感感染的小鼠气管。描述的 2 p-病媒模型将是相关的研究免疫细胞动力学在呼吸道感染。
最近, 在气道细胞动力学的可视化的几个模型被开发了9,10,11,12,<sup class="x...

双光子活体显微术 (2 p-病媒) 是一种有效的技术, 实时成像细胞间的相互作用, 因为它们发生在他们的自然环境1。这种方法的一个主要优点是, 它允许在更大的试样深度 (500 µm 到1毫米) 的细胞过程研究与其他传统成像技术2相比。同时, 两光子激光器产生的两个低能光子的使用最大限度地减少了通常与图像采集过程2相关的组织光损伤。在过去的十年中, 2 p-病媒应用于研究不同类型的细胞相互作用的几个学科3,4,5。这些研究特别相关的免疫细胞, 其特点是其高活力和形成明显的接触后, 其他细胞和环境产生的信号。2对病原体与宿主6之间的相互作用也进行了研究。事实上, 以前已经表明, 一些病原体可以改变免疫细胞之间接触的类型和持续时间, 从而妨碍免疫应答7。
气道粘膜是第一个场所, 其中免疫反应的空气病原体产生8。因此,在体内分析这种组织中的病原宿主相互作用, 对于了解宿主防御机制在感染过程中的启动至关重要。然而, 2 的气道病媒是挑战, 主要是由于动物的呼吸周期产生的文物, 这破坏了图像采集过程。最近, 不同的手术模式被描述为成像小鼠气管9,10,11,12和肺部13,14,15, 16。气管 2 p-病原学模型代表了一个良好的设置, 以可视化的初期阶段的免疫反应在上呼吸道, 而肺肺泡 2 p-病原细胞模型更适合研究晚期感染。肺模型的存在与充气肺泡的存在有关, 限制了激光的光穿透, 使肺内呼吸道的黏膜层无法在体内成像17.反之, 由连续上皮形成的气管结构有利于图像的采集。
在这里, 我们提出了一个协议, 其中包括详细说明了执行流感感染所需的步骤, 动物的外科准备, 和2对气管的 p 病媒。此外, 我们描述了一个具体的实验设置, 以可视化中性粒细胞和树突状干细胞 (DC), 两种免疫细胞类型, 发挥重要作用的调解机制, 以抵御流感病毒18,19.最后, 我们描述了一种分析中性粒细胞-DC 相互作用的过程。这些接触已经显示, 以调节 DC 激活, 并随后, 影响免疫反应的病原体20。

<table>
	<tbody>
		<tr>
			<td>Gigasept instru AF</td>
			<td>Sch&#252;lke &#38; Mayr GmbH</td>
			<td></td>
			<td>4% solution</td>
		</tr>
		<tr>
			<td>CD11c-YFP mice</td>
			<td>Jackson Laboratories</td>
			<td>008829</td>
			<td>mice were bred in-house</td>
		</tr>
		<tr>
			<td>CK6-ECFP mice</td>
			<td>Jackson Laboratories</td>
			<td>004218</td>
			<td>mice were bred in-house</td>
		</tr>
		<tr>
			<td>1 X Dulbecco&#39;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10 X Dulbecco&#39;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td>
			<td>Sigma</td>
			<td>D1408-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>Sigma</td>
			<td>E0414-1L</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Ketamin Labatec</td>
			<td>Labatec Pharma</td>
			<td>7680632310024</td>
			<td>Store at RT, store at 4&#176;C when in solution of ket/xyl mixture</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazin)</td>
			<td>Bayer</td>
			<td>6293841.00.00</td>
			<td>Store at RT, store at 4&#176;C when in solution of ket/xyl mixture</td>
		</tr>
		<tr>
			<td>26 G 1 mL Sub-Q BD Plastipak</td>
			<td>BD Plastipak</td>
			<td>305501</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G 0,3 mL BD Micro-Fine Insulin Syringes</td>
			<td>BD</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 40 &#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Syringes</td>
			<td>BD Plastipak</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Microlance 3 18 G needles</td>
			<td>BD</td>
			<td>304622</td>
			<td></td>
		</tr>
		<tr>
			<td>Introcan Safety 20G (catheter)</td>
			<td>Braun</td>
			<td>4251652.01</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Cluster</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 + HEPES (1X)</td>
			<td>ThermoFisher Scientific</td>
			<td>42401-018</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Liberase TL Research Grade</td>
			<td>Roche</td>
			<td>5401020001</td>
			<td>Store at -20&#176;C / collagenase (I and II) mixture</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Amresco (VWR)</td>
			<td>0649-50KU</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>CellTrace Violet stain</td>
			<td>ThermoFisher Scientific</td>
			<td>C34557</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>PE-10 Micro Medical Tubing</td>
			<td>2Biological Instruments SNC</td>
			<td>#BB31695-PE/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Plastic Tape</td>
			<td>M Plast</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Plasticine</td>
			<td>Ohropax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Tolerance Glass Coverslip 15mm Round</td>
			<td>Warner Instruments</td>
			<td>64-0733</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Portable Animal Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuvo Lite mark 5</td>
			<td>GCE medline</td>
			<td>14111211</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniTag (gaseous anesthesia and heating bench)</td>
			<td>Tem Sega</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SURGICAL BOARD</td>
			<td>University of Bern</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrimScope II Two-photon microscope</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Vision Ti:Sa lasers</td>
			<td>Coherent Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25X NA 1.05 water immersion objective</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr>
			<td>The Cube&#38;The Box incubation chamber and temperature controller</td>
			<td>Life imaging Services</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 9.1.0</td>
			<td>Bitplane</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td>Statistical software</td>
		</tr>
	</tbody>
</table>

imaging cell interaction in tracheal mucosa during influenza virus infection using two-photon intravital microscopy

细胞和细胞-病原体相互作用的分析是了解感染免疫反应动力学的重要工具。双光子活体显微镜 (2 p-病媒) 允许观察活体动物深层组织中的细胞相互作用, 同时尽量减少在图像采集过程中产生的漂白。迄今为止, 对2种对淋巴和非淋巴器官的 p 病媒进行了不同的模型描述。然而, 由于与动物呼吸循环有关的运动, 呼吸器官的成像仍然是一个挑战。
在这里, 我们描述了一个协议, 以可视化在体内免疫细胞相互作用的小鼠的气管感染流感病毒使用 2 p-病媒。为此, 我们开发了一种自定义成像平台, 包括气管的手术暴露和插管, 其次是获取粘膜上皮中中性粒细胞和树突状细胞 (DC) 的动态图像。此外, 我们详细的步骤, 以执行流感鼻腔感染和流动细胞分析的免疫细胞在气管。最后, 分析了中性粒细胞和直流运动及其在电影过程中的相互作用。该协议允许生成稳定和明亮的4D 图像, 用于评估气管内细胞间的相互作用。

在这项工作中, 我们描述了一个详细的协议来研究体内的运动和中性粒细胞和 DC 之间的相互作用, 在流感感染的小鼠气管 (图 3A)。为此, 我们分离 CFP+中性粒细胞 (92% 纯度;图 3B)从 CK6-ECFP 鼠, 我们过继转输转移到 CD11c-YFP 鼠感染流感。在那之后, 我们在3天的私家侦探身上执行了2的气管内病媒。此时, 我们观察到感染?...

Watch this Scientific Journal Video about Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy at JoVE.com

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

在本研究中, 我们提出了一个协议, 执行双光子活体成像和细胞相互作用分析的小鼠气管粘膜感染后流感病毒。这个协议将是相关的研究人员研究免疫细胞动力学在呼吸道感染。

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

imaging-cell-interaction-tracheal-mucosa-during-influenza-virus

Research

JoVE Journal

Immunology and Infection

8.2K 次观看.  Università della Svizzera italiana (USI). 在本研究中, 我们提出了一个协议, 执行双光子活体成像和细胞相互作用分析的小鼠气管粘膜感染后流感病毒。这个协议将是相关的研究人员研究免疫细胞动力学在呼吸道感染。

视频: Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

双光子激光显微镜在细胞膜修复中的应用

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.