我们要感谢马丁、Nobes、理查森和伍德实验室的成员进行有益的讨论。我们还感谢沃尔夫森 Bioimaging 设施 (英国布里斯托尔大学), 布卢明顿证券中心 (印第安纳大学, 美国) 和维也纳果蝇资源中心 (果蝇种群) 和 Flybase (最新果蝇基因注释)。这项工作得到了一项由湄公河和水 (MR/J002577/1) 的项目补助金, 一个水的威康信托高级研究员和一个威康信托调查员奖给下午。

该协议由四个主要的顺序步骤组成: (1) 果蝇种群的制备和果蝇蛹的分期, (2) 蛹解剖和安装, (3) 蛹伤, (4)在体内时间推移共焦成像。
1.果蝇种群的制备和蛹的分期
<ol><li>获得适当的果蝇种群 (见介绍和材料表)。</li>
	<li>收集适当基因型的年轻健康成年蝇。<ol>
<li>选择成年苍蝇使用二氧化碳气垫简要麻醉苍蝇和罚款画笔转移适当的基因型或性别的苍蝇的收集瓶。</li>
		<li>添加20处女女性和20男性到每个瓶子包含标准的飞行食物媒介 (玉米粉-糖蜜-琼脂混合物, 看见材料表) 补充与酵母。</li>
	</ol>
</li>
	<li>对于最佳蛹世代, 提示成人每天飞行到新的食物在新鲜的小瓶, 保持所有小瓶在25°c。 注: 如果 Gal4-UAS 系统31被用于驱动谱系特异基因表达, 所有步骤应执行在25摄氏度或以上, 因为 Gal4-UAS 系统是温度敏感。</li>
	<li>18 h 在预定成像会话之前, 从瓶子中选择至少10新形成的白色前蛹 (图 1A), 使用镊子或细画笔将蛹从内瓶表面取出, 然后从瓶中 (即蛹壳形成后0小时)把蛹小心地转移到一个干净的空塑料瓶的一侧。 注: 流浪 3路龄幼虫向上爬出食物培养基, 接受蛹;新形成的白色预蛹很容易辨认, 因为他们拥有外翻前气孔和是固定的 (不像 3rd龄幼虫)。角质层转变成 "蛹壳" (蛹的情况), 最初是软的和白色的17。必须注意避免损害蛹, 因为这不仅会导致不必要的伤害引起的炎症反应, 而且还有一个重大的发展延迟。</li>
	<li>年龄选定的蛹到适当的发展阶段 (18 h APF 将给最佳的结果) 在小瓶在25°c。 注意: 随着蛹的发育, 蛹的情况将逐渐变暗, 更加脆弱。</li>
	<li>提前准备下一步的其他试剂。要制造庚烷胶, 将20厘米长的卷起双面胶带与20毫升庚烷在50毫升离心管中, 在室温下用石蜡膜和岩石密封在工作台上过夜。</li>
</ol>2.果蝇蛹的制备和解剖
<ol><li>将舞台上的果蝇蛹转移到安装在玻璃滑梯上的双面胶带上。定位蛹, 使腹侧牢牢粘在胶带上, 背部侧朝上 (图 1B)。 注意: 蛹的前部将从蛹的前端突出的两个气孔中辨认出来。</li>
	<li>用镊子和微剪刀在 brightfield 解剖显微镜下, 小心地从保护蛹壳套管中取出蛹 (图 1B-D)。<ol>
<li>最初, 用镊子在蛹壳的最前面的区域做切口 (图 1B)。确保这个地区的蛹病例是空心的, 缺乏蛹组织, 因为蛹在早期蛹发育过程中会收缩。</li>
		<li>在最初的切口后, 用镊子或 microscissors (图 1C) 仔细地撕开或切开蛹的前向后方向, 直到蛹完全游离于褐色不透明的脆性套管 (图 1D)。 注意: 在这个阶段蛹是非常脆弱的, 必须注意避免刺穿蛹表面;穿刺很明显, 血淋巴迅速从穿刺部位漏出。有穿刺的蛹, 无论多么小, 都应该丢弃。</li>
	</ol>
</li>
	<li>用庚烷胶在玻璃底盘中安装蛹。<ol>
<li>使用20毫升吸管尖端放置10毫升前准备的庚烷胶 (见上文1.6 节) 在玻璃底菜线上。</li>
		<li>在用镊子将解剖过的蛹小心地转移到庚烷胶上之前, 允许胶水干燥5秒 (图 1E)。</li>
		<li>为了容易受伤和成像, 线的蛹在一排;建议大约5蛹, 但更多的将是可管理的经验。 注: 在使用直立成像系统时, 建议使用庚烷胶, 但在使用倒置系统时并不必要。如果胶水在成像过程中产生光学畸变, 蛹可以直接放在 coverglass 上--蛹组织和玻璃之间的天然黏附力在大多数情况下都足够稳定成像, 只要在移动成像时需要注意。在显微镜之间的盘子。</li>
	</ol>
</li>
	<li>为了获得最佳效果, 芒上蛹, 使机翼在 coverglass 上平坦, 机翼表面大部分与 coverglass 直接接触 (图 1F)。用镊子将蛹卷起来, 以改变其位置, 以确保机翼安装正确。</li>
	<li>为防止在成像期间的样品脱水, 在安装结束时, 添加一条浸泡在蒸馏水中的吸水过滤纸, 以避免扰乱蛹 (图 1E)。把盘子盖上盖子。 注意: 蛹现在已经准备好受伤和成像。</li>
</ol>3. 激光致果蝇蛹翼伤
<ol><li>将含有安装蛹的玻璃底盘转移到宽场显微镜上, 配备可调谐激光消融系统。<ol>
<li>使用脉冲紫外线空气冷却氮气泵浦激光调谐到 435 nm-见资料表, 详细11;照明用的光的精确波长由用户通过适当的染料细胞选择。</li>
	</ol>
</li>
	<li>使用 brightfield 光学, 调整显微镜阶段控制, 以找到第一个蛹的蛹机翼受伤 (图 1F)。</li>
	<li>为获得最佳结果, 使用油浸泡40X 或63X 物镜进行成像和激光消融;确保所使用的浸入式液体 (油或甘油) 在消融和成像系统之间是一致的。调整显微镜, 以聚焦在蛹翼上皮的平面上, 最接近玻璃盖玻片 (即重点在上皮的区域受伤) 使用精细对焦控制旋钮。</li>
	<li>使用显微镜阶段调整, 定位蛹机翼, 使受伤地区直接与已知靶区的消融激光。</li>
	<li>使用能量密度衰减器幻灯片安装在显微镜上, 手动调整烧蚀灯的功率水平。 注: 衰减器滑块有点击停止和裁决, 以确定衰减的相对水平, 并允许使用可重现的设置。</li>
	<li>使用外部手动触发控制, 激活烧蚀激光器, 使用一个简单的点击触发器来制造伤口。检查烧蚀部位瞬态气泡的外观, 因为伤人通常会伴随它。检查激光诱导伤是否成功使用适当的荧光过滤器可视化蛹上皮。 注意: 小心避免意外暴露在激光束上, 因为光束反射会引起严重的眼睛或皮肤损伤。</li>
	<li>如果伤人不成功, 改变焦距平面 (将显微镜的焦点稍稍移动到当前焦距的上方或下方), 然后重复一次点击消融扳机。或者, 使用衰减器滑动逐渐增加激光功率, 直到达到所需的伤口尺寸。</li>
	<li>为了改变烧蚀激光脉冲重复率, 使用后控制面板上的重复率旋钮 (它将速率从小于1的脉冲/秒改为60赫兹)。对于最佳伤人, 设置脉冲重复率为40赫兹。</li>
	<li>要产生不同大小的伤口, 使用能量密度衰减器滑动来手动调整烧蚀光的功率水平。</li>
	<li>避免伤害所有安装的蛹, 并使用这些未烧蚀的蛹作为未受伤控制。</li>
	<li>为保持一致的结果, 定期调整消融系统 (使用相关的操作手册)。同时, 清洗并重新填充控制激光输出波长的染料谐振器单元。</li>
</ol>4.体内时间失效共焦成像
<ol><li>快速将玻璃底盘转移到适当的显微镜上进行定时成像。 注: 为了获得最佳效果, 请使用高规格的共焦或旋转圆盘显微镜, 配备灵敏的探测器, 可以检测 GFP 和 mCherry 显影。</li>
	<li>要图像整个蛹机翼, 使用低放大倍数 (如20X) 物镜 (图 2A)。为了图象创伤修复和伴随的炎症反应以高空间分辨率, 使用油浸泡 40X (na 1.3) 或 63X (na 1.4) 物镜 (参见图 2BD的代表性图)。</li>
	<li>打开与显微镜相关的适当的图像捕获软件。</li>
	<li>使用图像捕获软件, 打开适当的激光器,如488 nm 和 561 nm 激光器, 以可视化 GFP 和 mCherry 显影, 分别 (通过点击相关框), 并调整激光功率和增益/偏移设置, 使充足的荧光信号, 同时避免像素饱和;使用尽可能低的激光功率 (在 5-20% 范围内), 以最小化漂白和毒性。</li>
	<li>为了捕捉修复上皮和炎症细胞的招募, 设置显微镜记录一个 z 栈使用微调旋钮在控制面板上;为获得最佳结果, 请设置软件 (使用手动按钮单击) 记录 z 切片通过蛹翼 (最小每3毫米), 从受伤的上皮顶部到下面的细胞外空间 (包含迁移包囊), 以实现一个大z 堆叠 (在 50-100 毫米的范围内)。</li>
	<li>对于时间推移成像, 记录 z 栈在固定的时间间隔 (最少每三十年代) 至少1小时后伤后。 注意: 所选 z 栈之间的确切时间间隔代表了捕捉快速变化的细胞动力学与避免样品的光漂白之间的权衡。</li>
	<li>为了同时图像多蛹 (包括未烧蚀的未受伤控制), 使用一个机动化的阶段 (连接到显微镜) 和多位置采集功能在成像软件。在软件中使用舞台位置控制旋钮手动设置每个蛹的位置, 然后为每个蛹手动设置相应的 z 堆栈限制 (顶部和底部)。</li>
	<li>使用 z 堆栈投影或3维渲染, 在图像捕获期间或以后使用专家图像分析软件 (如 ImageJ32) 可视化时间推移图像。例如, 要跟踪单个包囊 (如图 2C'和D ') 的移动, 请使用开放访问 ImageJ 插件 "TrackMate" 或 "手动跟踪" (在33中发布的方法) 跟踪全血细胞原子核,34)。</li>
	<li>使用 photoconvertible 探针 (如枫30) 在成像过程中选择性地 photoconvert 和标记上皮或免疫细胞的子集。<ol>
<li>在成像软件中打开适当的模块以执行 photoconversion (如酶、荧光恢复后的光漂白), 并激活405nm 激光 (通过单击相关软件框)35。</li>
		<li>使用正方形、圆形或手绘选择工具选择要在酶软件中 photoconverted 的单元格。在酶软件中, 将 photoconversion (漂白) 的时间路线设置为单个迭代/帧, 并将 405 nm 激光器设置为20% 激光功率。手动单击 "启动实验" 以执行 photoconversion。</li>
		<li>退出酶模块 (单击 "关闭") 并返回到软件中的原始图像屏幕;使用 488 nm 和 561 nm 激光器, 通过设置 z 堆栈和时间推移记录, 以图像的 photoconverted 和非 photoconverted 细胞的行为。 注意: Photoconverted 探头在初始 photoconversion 后的许多小时保持稳定, 使 Photoconverted 细胞的行为能够随时间推移 (至少5小时)。例如, 伤口中的炎症细胞可以有选择地 photoconverted (图 2F), 当它们从受伤部位 (图 2G 和 H) 解决时, 它们的行为就会随之而来。</li>
	</ol>
</li>
</ol>

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作者声明他们没有竞争利益冲突。

急性炎症反应的组织损伤是一个复杂和高度动态的过程, 这是必不可少的, 以协调修复受伤的组织, 包括清除坏死碎片和打击感染。为了充分理解和解开这一反应的基本方面, 重要的是在体内对3维活体样本进行研究, 以使所涉及的各种细胞血统的精确行为和相互作用准确地随着时间的推移。对这些细胞动力学进行实时分析, 可以用经典的免疫组化技术, 比固定样本的静态单时间点更详细地描述突变体表型。传统上, 大多数使用基因处理果蝇模型的活体成像研究都使用了果蝇发育的胚胎阶段, 因为它的光学半透明性和不动性与后期发育阶段相比4 。,5. 然而, 最近, 我们的小组和其他人已经开发了果蝇蛹作为一种新的模式, 以执行高分辨率和长期的创伤修复和炎症的慢性影像在体内8,22 ,23。这种新兴的方法为解开炎症细胞行为的基本方面提供了令人振奋的长期潜力, 可以进一步适应研究其他细胞血统的动态行为 (如果蝇脂肪体38) 组织损伤后。
有许多关键因素在准备和成像受伤的果蝇蛹, 这将决定质量的成像结果所述。可以说, 最困难的步骤是描述的协议是仔细解剖和精确定位的蛹之前, 伤和成像。蛹在这个发育阶段是极其脆弱的, 甚至小的意外伤害的蛹在准备阶段将显著损害实验;任何可能持续性损伤的蛹都必须从实验中丢弃, 因为这种损伤可能激活其自身的炎症反应, 这可能会导致对其他地方的炎症细胞行为产生更广泛的 (甚至系统性的) 影响。蛹。由于在这些实验中使用的蛹的持续发展 (正在进行大量的组织重排以准备成年的组织), 偶尔蛹会在成像过程中移动。然而, 如果蛹没有正确安装在与 coverglass 直接接触的机翼 (或其他组织的平坦表面) 上, 蛹的滚动就更有可能发生;使用庚烷胶稳定盖玻璃上的蛹, 应尽量减少这种不良的运动。因此, 在显微镜之间移动样品时, 也必须非常小心避免逐出的蛹从它们精心排列的位置上移出;理想情况下, 伤激光将被连接到同一显微镜, 用于随后的时间推移成像和照片转换。
除了蛹解剖和安装步骤的熟练程度, 使用的果蝇蛹的确切基因型将对所产生的成像数据的质量产生重大影响。例如, 在一个单独的蛹基因型中, Gal4驱动程序和无人驾驶的结构 (例如, 枫) 的拷贝数量将在随后的成像过程中确定信噪比。通常情况下, Gal4或无人管的结构的拷贝越多, 组织内荧光蛋白 (如GFP 或枫) 的总水平就越大。然而, 荧光蛋白的最佳水平将是一个谨慎的平衡, 以提高组织荧光足以使高质量的成像 (使使用较低的激光功率, 减少漂白和成像延长时间期), 但不引起荧光诱导的细胞毒性;在每个实验中, Gal4 和无人参与结构的最佳数目将根据特定的驱动程序和显影的不同而有所不同。应注意提高蛹在25°c (或以上, 高达29°c), 因为 Gal4-UAS 系统是温度敏感的, 将无效的低温31。为了达到对Gal4驱动表达的组织或时间特异性的额外控制水平, Gal4-UAS 系统阻遏 Gal80 也可以包括在蛹基因型39中。Gal80 可以用来抑制特定组织内的 Gal4 活动 (使用组织特定的 Gal80) 或在特定时间 (使用温度敏感 Gal80)。Gal4-UAS 系统可以与其他独立的二进制系统 (如莱克萨lexAop和QUAS系统) 进一步结合, 生成具有多个构造的果蝇(如显影、rna 干扰线或其他基因构造) 同时表达在不同组织的范围内39。
使用这种新的果蝇蛹模型提供了一些优势优于传统的胚胎方法。与15期胚胎 (大多数胚胎损伤研究的阶段) 相比, 短期成像 (最多3小时), 蛹可以在很长一段时间内被成像 (主要是在蛹发育96小时后的成年后)。).此外, 包囊 (果蝇先天免疫细胞) 在蛹组织 (和可用于影像学) 中存在的数量远多于胚胎中存在的数量有限, 这使我们能够聚集更多使用相同的样本总数对全血细胞行为的成像数据。关键的是, 这反过来, 使我们能够应用更复杂的数学建模来分析包囊行为, 并提取伤口引诱和炎症反应的新特征, 否则将保持实验性无法访问23。蛹模型的另一个优点是, rna 介导的基因击倒比早期胚胎阶段效率要高得多, 允许用 Gal4-UAS 系统等二进制系统改进组织或特定时间基因失活的分析。39. 在这一阶段, rna 干扰的效率使我们有可能执行大规模的 (甚至是无偏见的全基因组) rna 干扰屏幕, 以寻找参与创伤修复或炎症细胞行为的新玩家。
然而,果蝇蛹显然不能用来研究由胚胎致死的基因突变造成的表型;因此, 对于胚胎发育至关重要的基因的功能性和活体成像研究, 必须在胚胎中进行, 除非 rna 介导的基因在时间或组织特定的方式下被击倒, 允许发展发生到蛹阶段。胚胎也仍然是研究和活图像的选择模型的免疫细胞行为的某些特征, 包括免疫细胞从其来源的发展扩散, 运动接触抑制和凋亡尸体吞噬在发育组织雕刻5,8尚未观察到的蛹模型中产生。虽然研究果蝇幼虫和成人提供了一个重要的洞察机制的基础伤口修复和炎症40,41,42,43,由于样本的固有流动性质, 在这些阶段进行的44种活体成像研究已被证明是困难的。虽然幼虫可以被麻醉, 允许短暂的活体成像, 由于麻醉的暂时性质, 只有短快照的活伤口修复或炎症反应可以可视化45。最近的一项研究已经制定了一个改进的协议, 允许长期成像的幼虫伤口愈合46, 虽然准备和成像仍然比在胚胎或蛹更具挑战性。从长远来看, 我们设想通过利用最适当的发展阶段来解决每个具体问题, 在所有四种不同的果蝇阶段进行研究-从胚胎到幼虫和蛹期到成年 (每个都有他们自己独特的优势和局限性)-将提供互补的洞察力的分子和细胞机制驱动伤口修复和炎症。
今后, 本议定书的创伤和长期成像果蝇蛹可以很容易地适应研究一系列的炎症相关的现象, 并具有深远的潜力, 以揭示新的特点炎症创面反应。长期成像的结合, 连同 photoconvertible 显影 (如枫) 的应用, 对于了解先天免疫细胞行为的动态具有重要的价值, 尤其是对伤口炎症反应。通过具体标记免疫细胞的个体或亚群 (如被招募到伤口的人), 就可以分析暴露在一个环境线索 (如尸体或伤害) 对免疫细胞随后的反应的影响。提示。果蝇包囊的炎症行为可以通过以往的经验来改变, 例如, 它们是在发育12期间对凋亡尸体的早期吞噬作用作出反应的, 但仍有待观察其他环境线索是否引发类似的启动事件。尽管迄今为止对蛹伤口的研究集中在先天炎症反应上, 蛹机翼模型也为活体图像和解剖上皮伤口修复机制提供了理想的机会。此外, 这种蛹成像方法也可以适应研究其他细胞谱系的动态行为, 以应对组织损伤38, 无论是在蛹机翼本身或其他容易接近的蛹组织 (如眼睛, 腿或胸部)。最后, 通过结合果蝇的遗传驯良和长期蛹成像的方便性, 新的上皮修复或炎症调节器可以通过应用无偏见的全基因组的击倒来发现方法。

有效和有效的炎症反应是任何有机体对抗感染, 清除碎片, 并安排修复受伤组织1的关键。虽然这种反应是大多数组织损伤的必然结果, 炎症需要严格的调节, 因为不适当的炎症反应已与各种不同的人类疾病 (包括慢性非愈合伤口) 联系在一起,疤痕过多, 易患癌症)1,2,3。鉴于这一临床相关性, 重要的是要获得一个更详细的了解的分子和细胞机制驱动炎症反应, 以制定新的预后指标和策略, 以治疗一系列慢性炎症条件, 这可能会保护修复组织免受长期和不必要的炎症。
近年来,果蝇已成为一个建立良好和有价值的模型系统, 以解剖的基本特征的炎症反应保存从昆虫到人类4,5。目前,果蝇提供了比目前在其他实验模型 (如小鼠或斑马鱼) 更大的遗传驯良, 允许在体内精确的时空遗传操作 (以灭活或在指定的发育时间点内, 在特定细胞类型中表达感兴趣的任何基因, 并能轻松地对全基因组屏幕6,7。传统上, 大多数活体影像学研究果蝇的伤口愈合和炎症是在胚胎阶段进行的, 因为胚胎是静止的 (不像果蝇幼虫或成人) 和光学半透明, 使无与伦比的高分辨率在体内成像8。这使得研究人员能够想象迅速而有力地招募果蝇先天免疫细胞 (包囊) 到伤口部位, 以应对机械或激光致胚胎上皮细胞损伤9,10,11,12,13,14. 通过将这些活体成像研究与基因操作结合起来,果蝇胚胎的研究发现了许多重要的免疫细胞蛋白, 可以控制体内的炎症细胞行为。例如, CED-1 同源德雷柏 (一种 ITAM 域含蛋白) 被确定为一种重要的 ' 损伤受体 ', 它以 H2O2依赖的方式将招募果蝇免疫细胞介导到伤害15。在免疫细胞内的德雷柏水平依次由钙诱导的 JNK 信号和凋亡尸体摄取的下游升高12。全血细胞运动还需要复杂的骨架改变, 以协调定向移向伤口, 这取决于骨架监管机构的活动, 如肌动蛋白捆绑的蛋白质 Fascin16 , 和一家小GTPases Rac 和9。
果蝇是一种 holometabolous 昆虫, 经过胚胎发育后的额外幼虫和蛹阶段, 在达到成年17之前。果蝇蛹已被开发成一个额外的模型, 为非侵入性活成像的各种动态细胞事件, 包括发育细胞迁移18, 细胞分裂19, 细胞生长20, 和肌肉收缩21。最近, 它已经建立了一个新的模型, 以研究创伤修复和炎症在体内22,23的动态。
正如在胚胎阶段,果蝇蛹是静止和光学半透明的, 经过仔细解剖, 从他们的不透明蛹病例18。通过利用这种光学透明性, 你可以遵循先天免疫细胞 (包囊) 的体内行为, 以应对果蝇蛹组织中的组织损伤, 如蛹翼22。蛹翼存在于一个简单的双层结构, 由两个大的扁平上皮片组成, 在机翼周围连接;这两个上皮层之间的细胞外空间充满血淋巴 (昆虫血) 和大量的运动包囊24。就像在胚胎中, 机械或激光导致的机翼上皮损伤触发迅速招募包囊到受伤地点23。然而, 这个蛹阶段提供了一些明显的优势, 在早期的胚胎阶段的成像。受伤的蛹可以成像在较长的时间段 (至少5小时), 更多的组织面积可用于实验性摄动 (如多发伤的产生), 并有大量的包囊目前在这个阶段 (在数学分析中提供更多的距离来提高统计能力的细胞轨迹)。此外, rna 介导的基因失活的效率在蛹阶段有很大的改善, 使得许多基因在组织中被 "击倒", 或者与较传统的胚胎整体突变方法相比更具时间特异性。
为了遵循这个新的蛹模型中的伤口再上皮和伴随的炎症反应的动力学, 必须标记两个不同的细胞群体: 蛹上皮和先天免疫细胞 (果蝇包囊)。有许多不同的标记 (材料表) 可以标记这两种不同的细胞群--标记的选择取决于要研究的特定过程。为了标记蛹上皮, 含有无所不在表达的 GFP 标记的 e-钙黏蛋白 (标签 adherens 连接) 的果蝇线通常用来表示细胞边缘的位置, 或者是 gfp 标记的肌动蛋白结合领域的膜突 (标签肌动蛋白细胞骨架), 以可视化的伤口边缘收缩肌动蛋白环和前沿突起。为了标记果蝇包囊, 全血细胞特定的srp-Gal425驱动核 RFP 的表达 (用于核追踪), 细胞质 gfp 或 gfp 标记膜突 (分别标记细胞质或肌动蛋白细胞骨架) 或使用 photoconvertible 荧光 (如枫)。事实上, 在组合中使用多个免疫细胞标记通常是有利的, 从而能够同时分析核运动和细胞形态 (见有代表性的结果)。然而, 由于这项议定书涉及果蝇蛹的使用, 只有遗传标记的组合是可行的, 直到中蛹阶段可以利用。而且, 胚胎致命的股票也不合适。这不太可能是一个问题时, 成像控制 (或野生型) 蛹, 但重要的是要考虑当基因被击倒或抗原, 以评估其对伤口闭合或炎症的影响。在基因击倒 (或过度表达) 导致的早期致死性的情况下, Gal80ts构造可以用来诱导 Gal4-driven 在以后的发展中被击倒 (参见讨论)。
在我们最近的研究中, 进入蛹阶段使我们能够收集足够的免疫细胞弹道数据, 用复杂的数学建模来分析炎症行为, 这反过来又让我们推断出了伤口的新细节。炎症引诱剂信号23。例如, 这种方法表明, 伤口趋化因子在发炎组织中缓慢传播200微米2/分钟, 比先前建议的小候选分子如 ATP 或 H2O2的速度要慢得多, 报告漫射26,27,28,29;这些小的 "损伤" 分子反而很可能充当宽松信号。此外, 通过遵循先天免疫细胞的长期行为, 因为他们解决从最初的伤口, 并暴露在第二 (在不同的 timepoints 后, 第一次), 我们发现了一个暂时的 ' 脱敏 ' 期间, 其中免疫细胞对随后的伤害视而不见23。利用蛹模型的长期成像潜能, 连同果蝇的遗传驯良, 你可以遵循特定的免疫细胞群体的行为 (如只有那些被招募到伤口部位的免疫细胞) 在对随后的侮辱的反应, 使用 photoconvertible 荧光30 , 可以完全表达在免疫细胞谱系23。
在这里, 我们描述了一个协议, 以可视化的创伤修复的动态和相关的炎症反应在高时空分辨率使用活果蝇蛹。我们提供了一个详细的方法, 以涵盖最初的蛹准备 (解剖和安装) 和随后的激光介导伤人和时间推移成像所需的步骤。我们还描述了使用可转换显影, 以允许在体内标记特定的免疫细胞子集。从长远来看, 我们设想这个新的果蝇蛹模型将打开令人兴奋的可能性, 以解剖复杂的信号动力学的基础上炎症反应组织损伤。通过应用更复杂的统计分析, 你可能发现反应的特点, 否则将保持实验性无法访问, 而改进的 rna 干扰效率可以在应用全基因组筛选免疫细胞在体内识别新的玩家调节免疫细胞的行为。

<table>
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ubiquitous GFP-tagged E-cadherin 
			;Ubi-p63E-shg.GFP; (chrII)</td>
			<td>Kyoto Stock Center, DGRC</td>
			<td>#109007</td>
			<td>Ubi-p63E promoter sequences drive expression of Drosophila E-cadherin (shotgun) tagged at the C-terminal end with GFP.</td>
		</tr>
		<tr>
			<td>ubiquitous GFP-tagged E-cadherin 
			;;Ubi-p63E-shg.GFP (III)</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#58742</td>
			<td>Ubi-p63E promoter sequences drive expression of Drosophila E-cadherin (shotgun) tagged at the C-terminal end with GFP.</td>
		</tr>
		<tr>
			<td>ubiquitous GFP-tagged Moesin 
			P{sGMCA}3.1</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#59023</td>
			<td>The ubiquitously expressed sqh promoter/enhancer drives expression of a fragment of Moesin (that includes the actin binding sequences) tagged with GFPS65T.</td>
		</tr>
		<tr>
			<td>hemocyte specific serpent-Gal4 driver 
			;srp-Gal4;</td>
			<td>Generated by Katja Bruckner</td>
			<td>Generated by Katja Bruckner</td>
			<td>Expression of Scer\GAL4 fused to a polyA tail is under the control of 2 genomic sequences from upstream of Drosophila serpent. Ref: Br&#252;ckner, K., Kockel, L., Duchek, P., Luque, C.M., R&#248;rth, P., Perrimon, N. The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell. 7 (1), 73&#8211;84, doi: 10.1016/j.devcel.2004.06.007 (2004).</td>
		</tr>
		<tr>
			<td>UAS-nuclearRFP 
			w1118;;P{UAS-RedStinger}6</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#8545 or #8547</td>
			<td>UAS regulatory sequences drive expression of the DsRed.T4 form of RFP which is tagged at the C-terminal end with a nuclear localisation signal</td>
		</tr>
		<tr>
			<td>UAS-cytoplasmicGFP 
			;;P{UAS-GFP.S65T}</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>Multiple stocks available (e.g. #1522)</td>
			<td>Expression of the S65T version of GFP by UAS regulatory sequences; the S65T variant exhibits increased brightness.</td>
		</tr>
		<tr>
			<td>UAS-photoconvertibleKaede 
			w1118;; P{UAS-Kaede.A}3</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#26161</td>
			<td>Kaede protein emits bright green fluorescence after synthesis, but changes efficiently to a bright stable red fluorescence on irradiation with UV.</td>
		</tr>
		<tr>
			<td>GFP-tagged spaghetti squash 
			w1118;;P{sqh-GFP.RLC}</td>
			<td>Bloomington Drosophila Stock Centre (Indiana University)</td>
			<td>#57145</td>
			<td>The sqh coding region, which is tagged at the C-terminal end with a T:Avic\GFPS65T tag, is expressed under the control of the natural sqh promoter.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ingredients for fly food media</td>
			<td></td>
			<td></td>
			<td>Fly food media is made according to standard procedures (see Greenspan, R. 1997. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Press. 1-191 pp.)</td>
		</tr>
		<tr>
			<td>maize</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>soya flour</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>malt extract</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>molasses</td>
			<td>Wild Oats, Bristol, UK (or equivalent supplier)</td>
			<td>Contact supplier direct</td>
			<td>organic</td>
		</tr>
		<tr>
			<td>Difco agar</td>
			<td>BD Biosciences, Fisher Scientific</td>
			<td>DF0142-15-2</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Sigma</td>
			<td>402907</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Nipagen</td>
			<td>Sigma</td>
			<td>79721</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Dried baker&#39;s yeast</td>
			<td>Redstar, Dutscher Scientific, UK LTD</td>
			<td>Redstar, Dutscher Scientific, UK LTD</td>
			<td>For preparation of fly food</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sample preparation and mounting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Fine sable paintbrush</td>
			<td>Daler-Rowney (or equivalent)</td>
			<td>#0 or 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific (or Fine Science Tools)</td>
			<td>NC9404145</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Glass bottomed dishes for imaging</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td>We suggest using 35mm petri dishes, with at least a 10mm Microwell, 0.085-0.13mm cover glass, uncoated. Dishes with larger microwells will enable increasing numbers of pupae to be mounted and imaged in a single experiment.</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma</td>
			<td>51730-5ML</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Double sided sticky tape (e.g. Scotch)</td>
			<td>Agar Scientific</td>
			<td>AGG263</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>50ml tube (for heptane glue)</td>
			<td>Falcon tubes from Fisher Scientific</td>
			<td>14-432-22</td>
			<td>For preparation of heptane glue</td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Agar Scientific</td>
			<td>AGL4244</td>
			<td>For dissection of Drosophila pupae</td>
		</tr>
		<tr>
			<td>Dissecting stereo microscope with brightfield</td>
			<td>Leica (or equivalent)</td>
			<td>M50</td>
			<td>For dissection of Drosophila pupae</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>John Weiss International</td>
			<td>103123</td>
			<td>Miniature Research Scissors (straight)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Laser ablation and imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitogen ablation laser</td>
			<td>Spectra-Physics (or Andor equivalent)</td>
			<td>Model VSL-337ND-S</td>
			<td>For wounding, this should be attached to a widefield imaging system</td>
		</tr>
		<tr>
			<td>Multilaser confocal laser-scanning microscope (CLSM)</td>
			<td>Leica (or equivalent)</td>
			<td>TCS AOBS SP8 or SP5-II attached to a Leica DMi8 inverted epifluorescence microscope (or equivalent)</td>
			<td>Ideally including a motorised stage for multi-site and &#39;mosaic&#39; scanning, plus &#8216;hybrid&#8217; GaAsP detectors (that offer much greater sensitivity and boosting of low signal)</td>
		</tr>
		<tr>
			<td>Environmental chamber</td>
			<td>Life Imaging Services (or equivalent)</td>
			<td>&#34;Microscope Temperature Control System&#34;</td>
			<td>Attached to Confocal microscope for temperature control during imaging</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FRAP software module</td>
			<td>Leica (or equivalent)</td>
			<td>CLSM FRAP software module</td>
			<td>For performing photoconversion of photoconvertible fluorophores such as Kaede</td>
		</tr>
		<tr>
			<td>ImageJ (image analysis software)</td>
			<td>National Institutes of Health (NIH)</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td>Schneider, C.A., Rasband, W.S., Eliceiri, K.W. &#34;NIH Image to ImageJ: 25 years of image analysis&#34;. Nature Methods 9, 671-675, 2012.</td>
		</tr>
		<tr>
			<td>ImageJ plugin &#34;Manual Tracking&#34;</td>
			<td>National Institutes of Health (NIH)</td>
			<td>https://imagej.net/Manual_Tracking</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ plugin &#34;TrackMate&#34;</td>
			<td>ImageJ, NIH</td>
			<td>https://imagej.net/TrackMate</td>
			<td>Tinevez, JY.; Perry, N. &#38; Schindelin, J. et al. (2016), &#34;TrackMate: An open and extensible platform for single-particle tracking.&#34;, Methods 115: 80-90, PMID 27713081</td>
		</tr>
		<tr>
			<td>Volocity (high performance 3D imaging software)</td>
			<td>Perkin Elmer</td>
			<td>Volocity 6.3</td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>IMARIS (image analysis software)</td>
			<td>Bitplane</td>
			<td>IMARIS for Cell Biologists</td>
			<td>For image analysis</td>
		</tr>
	</tbody>
</table>

long-term in vivo tracking of inflammatory cell dynamics within drosophila pupae

在对组织损伤的快速炎症反应中, 先天免疫系统细胞迅速被招募到受伤部位。一旦在伤口上, 先天免疫细胞就会执行一些基本功能, 如抗感染、清除坏死碎片和刺激基质沉积。为了充分理解调节这种免疫应答的各种信号事件, 重要的是观察在体内的多细胞谱系 (和相互作用之间发生) 的复杂行为, 并在实时, 与高时空分辨率。果蝇胚胎的光学半透明性和遗传驯良已经建立果蝇作为一种无价的模式来生存图像和解剖炎症细胞行为的基本方面, 包括机制发展扩散, 清除凋亡的尸体和/或微生物病原体, 并招募伤口。然而, 最近的研究表明, 在果蝇生命周期 (果蝇蛹) 中使用更晚的阶段提供了许多明显的优势, 包括改进的 rna 干扰效率、更长的成像周期和显著增强的免疫细胞数。在这里, 我们描述了一个成像伤口修复的协议和相关的炎症反应, 在高时空分辨率的活果蝇蛹。为了遵循重新上皮和炎症的动态, 我们使用了一些特定的活体荧光标记物, 用于上皮和先天免疫细胞。我们还展示了可转换显影的有效性, 如枫, 用于跟踪特定的免疫细胞子集, 追踪他们迁移到受伤部位的行为, 并从中解决。

本协议描述了果蝇蛹的制备, 用于活体时移成像的创伤修复和体内炎症。使用这种方法, 应该可以产生多个高分辨率电影的蛹伤口闭合和炎症细胞招募轻松和图像的蛹长时间 (至少5小时) 伤后没有明显的漂白。
果蝇蛹制备的一般方案
图 1说明了在活体成像中制备果蝇蛹的最佳方法。果蝇白色 ' 预蛹 ' 收集在 ' 0 小时 ' 后, 蛹壳形成 (APF), 因为爬行幼虫停止运动, 并采取定型的蛹形状与外翻呼吸附件 (气孔) 可见在他们的前-最末端 (图 1A)。白色 0 h APF 预蛹允许开发18小时在25摄氏度, 蛹壳已经变成褐色 (图 1B), 然后用细钳和/或 microscissors 解剖 (图 1C, 删除保护蛹案件), 以揭示光学半透明蛹内 (图 1D)。解剖后, 蛹的翅膀将在蛹胸部的侧面可见 (在蓝色,图 1C-D) 的轮廓。在这个阶段, 其他蛹身体部位也很容易辨别, 包括眼睛, 腿和腹部 (标记在图 1D)。腿部也适用于伤口炎症研究, 并可使用上述相同的激光方法受伤。多 18 h APF 蛹可以同时安装在成像盘内 (图 1E, 使用庚烷胶和水浸泡滤纸, 以尽量减少脱水) 与平坦部分的机翼 (概述蓝色) 接触盖玻片 (图 1F);这是特别有利的, 如果成像显微镜配备了一个机动的阶段, 允许几个蛹记录在一个单一的成像周期。任何在解剖或安装过程中受损的蛹都应丢弃 (如蛹位于序列中的第三个,图 1E中的箭头)。其他身体部位 (如眼睛和腿, 轮廓粉红色) 也可能与盖玻片联系, 可用于伤人和随后的影像学 (图 1F)。在研究单个伤口的实验中, 激光诱导的损伤通常最好集中在机翼 (星号,图 1F) 上, 但如果要研究多处伤口, 还可以使用其他位置。
无菌伤人在果蝇蛹翼激活一个强健的炎症反应
为了跟踪创面修复和体内炎症反应,采用共聚焦时间推移显微镜 (图 2A-H) 对18h 型有源性果蝇蛹的受伤机翼进行了成像。在这个蛹阶段, 机翼上皮是简单的扁平细胞 (标记在这里使用 GFP 标记 e-钙黏蛋白标记单个细胞边界, 翼缘在图 2A中概述)。即使在未受伤翼 (低放大率,图2A 和 2A '), 大量的迁徙先天免疫细胞 (包囊, 标记使用srp-Gal4驱动细胞质 GFP, 绿色, 核 RFP, 洋红) 在血淋巴中发现 (昆虫血) 占据细胞外空间 (图 2A和2A)。激光致蛹翼上皮细胞损伤 (白色轮廓,图 2Bd) 刺激包囊快速迁移到伤口部位 (图2BD和免疫细胞核在图 2B'-D ')。用核标记 (如核 RFP ' 红刺 ') 与细胞质或骨架标记 (如细胞质荧光蛋白或 gfp 标记的膜突) 进行标记包囊特别有利, 因为它允许同时跟踪全血细胞核 (用于全血细胞行为的自动分析,如迁移速度和方向性) 和全血细胞形态学的可视化。后者是重要的, 因为它允许我们确定是否包囊是吞噬坏死的碎片 (图 2C, 嵌入) 在伤口边缘 (或在感染情况下的微生物)12,23 , 也允许我们遵循他们的突出行为, 当他们向或远离伤口的时候, 在它们的前缘伸展出精细的丝状伪足或 lamellipodia。
用适当的图像分析软件对全血细胞核轨迹进行简单跟踪32 (材料表) 显示了炎症反应的复杂时空动态, 类似于以前报告的受伤的胚胎9,36 (多色轨道,图 2C' 和D ')。在仅仅30分钟的伤害, 包囊位于最接近受伤地点开始定向移向伤口 (图 2C)。然而, 随着受伤时间的增加, 包囊位于逐渐远离受伤部位的地方也开始向伤口方向迁移 (图 2D)。这样, 就能观察到从伤口边缘向外扩散的免疫细胞反应的 "波", 我们设想将伤口趋化蛋白的扩散从损伤部位反射出去。这些时空免疫细胞动力学提供了一个有用的出发点, 我们最近的研究, 采用复杂的数学建模 (贝叶斯推理) 分析, 以推断新的性质的创伤引诱信号 (详细的方法出版于23)。通过对我们的计算模型 (将伤口引诱剂梯度与全血细胞偏倚) 进行校准, 以适应体内免疫轨迹的观察, 可以从我们的全血细胞弹道中提取伤口引诱剂的详细特征。数据 (如信号扩散率、源和信号产生的持续时间)23。
此外, 通过使用srp-Gal4 (绿色,图 2E) 在免疫细胞谱系中驾驶 photoconvertible 荧光枫的表达, 我们也能够选择性地标记这些迁徙翼包囊的亚群 (如被招募到伤口部位的人;E, 在紫外线诱导的 photoconversion 和 F, 后 photoconversion)。然后, 我们可以遵循这些包囊的行为, 随着时间的推移 (洋红,图 2G-H), 因为他们解决远离伤口部位 (箭头,图 2G和H), 并比较他们的行为是如何不同于那些非 photoconverted无法到达受伤部位的细胞。我们使用这种活体标记技术来证明, 包囊被招募到最初的伤口是暂时麻木的第二个伤口产生90分钟后23, 虽然这种差异标记技术也可以将来还有许多其他有见地的应用 (见讨论)。
使用这种方法, 我们还可以同时跟踪伤口修复或 epithelialisation 的时空动态 (图 2B-D)。在这里无所不在表达了 GFP 标记的 e-钙黏蛋白标签的细胞 adherens 结在整个机翼上皮, 并允许个别上皮细胞的形状, 随着时间的推移。伤口边缘很容易被识别为完整的 GFP 标记上皮细胞和未标记碎片 (白色虚线,图 2B-D) 之间的连接。对于大多数伤口, 伤口开始重新 epithelialize 在1小时的伤害和伤口边缘向前推进 (图 2D);这种大小的伤口通常会在 2-3 小时的伤害中愈合23。在胚胎和蛹的伤口闭合是由一个肌动球蛋白收缩电缆和领先的边缘肌动蛋白丰富的丝状伪足23,37驱动;这些骨架动力学可以直接可视化在伤口修复使用适当的活体记者, 如 gfp 标记的意大利面条南瓜 (肌球蛋白调节灯链) 或 gfp 标记的肌动蛋白结合膜突23。然而, 对于一些特别大的伤口, 肌动球蛋白电缆和前缘丝状伪足不坚持-这些伤口变得 ' 慢性 ', 从来没有完全重新 epithelialize23甚至在更长的时间 (超过24小时) 的体内成像。有趣的是, 与这些非愈合伤口相关的炎症反应明显不同于与正常急性创面相关的23提示异常免疫细胞行为可能是临床上有用的预后标志。.将来, 在蛹模型中使用长期成像 (超过24小时) 的慢性伤口的活体分析, 可能会对这种衰弱的状况提供重要的机械洞察力。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57871/57871fig1.jpg"> 图 1:果蝇蛹的准备为创伤和活成像。(A)果蝇白色预蛹收集在 0 h APF, 前端由外翻呼吸附属物 (气孔) 指示。(B) 在提高白色 0h APF 预蛹18小时25摄氏度后, 蛹壳呈褐色。蛹病例的解剖应始于最前面的区域 (箭头), 由外翻气孔指示, 因为蛹适当的是没有从这个地区, 和罚款钳和/或 microscissors 用于清除保护性蛹案件 (C)。蛹的翅膀将在蛹胸部的侧面可见 (蓝色轮廓)。(D) 蛹的病例完全从 18h APF 蛹中除去, 这里的蛹已被轧向90°显示侧面, 机翼被成像在蓝色的轮廓。(E F)五18h 有源 APF 蛹安装在玻璃盖玻片的成像盘内使用庚烷胶, 用水浸泡过滤纸, 以尽量减少脱水 (E)。蛹的第三个在序列 (箭头) 应该被丢弃, 因为损坏发生在准备过程中。蛹是安装在与盖玻片 (f) 接触的机翼的平坦部分 (概述蓝色) 和激光诱发的伤口集中在机翼 (星号, F), 虽然其他地点可能会使用, 如果多个伤口是进行研究。在 (D) 的图象由织布工等的允许适应, 201623。<a href="https://www.jove.com/files/ftp_upload/57871/57871fig1large.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/57871/57871fig2.jpg"> 图 2: 动态体内分析炎症反应对组织损伤的影响.从保护性蛹病例解剖的蛹和安装在玻璃盖玻片 (A) 受伤, 随后成像使用共焦时间推移显微镜 (B H)。未受伤蛹翼的低放大视图 (a, 白色轮廓的翼缘), 包含大量的迁徙包囊 (a')。激光诱导损伤蛹翼上皮细胞 (B D,细胞边界标记使用 GFP 标记的果蝇e-钙黏蛋白; 伤口边缘白色概述) 激活多个包囊的迁移快速炎症反应 (srp-Gal4驱动表达核 RFP, 洋红和细胞质 GFP, 绿色) 对伤口部位 (B D; 有代表性的帧从一个时间推移电影, 其中每帧是投影25片3微米每)。手动跟踪全血细胞轨迹 (多色轨道、 C '和D ') 表明炎症反应的复杂时空动态, 类似于受伤胚胎的报告。包囊也吞噬坏死的细胞碎片在伤口部位 (箭头, C和也插入)。photoconvertible 荧光枫在免疫细胞谱系中的表达 (绿色, e, 使用srp-Gal4) 使伤口吸收包囊 (箭头, e) 有差异标记 (洋红, F), 并遵循随着时间的推移, 他们从伤害站点 (箭头, G和H) 解决。采用以下蛹基因型: (A D) w1118;ubi-gfp, srp-Gal4 > 无人技术-gfp (II);nRFP (III)和 (E H) w1118; Gal4 (II);枫 (III)。图片适应了由织布工等的允许, 201623。<a href="https://www.jove.com/files/ftp_upload/57871/57871fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae at JoVE.com

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

在这里, 我们提出了一个实时成像伤口修复的协议和相关的炎症反应在高时空分辨率的体内。该方法利用果蝇发育的蛹阶段, 实现对特定细胞种群的长期成像和跟踪, 并与有效的 rna 干扰介导的基因失活相兼容。

果蝇蛹中炎症细胞动力学的长期体内追踪

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

long-term-vivo-tracking-inflammatory-cell-dynamics-within-drosophila

Research

JoVE Journal

Immunology and Infection

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

8.7K 次观看.  University of Bristol. 在这里, 我们提出了一个实时成像伤口修复的协议和相关的炎症反应在高时空分辨率的体内。该方法利用果蝇发育的蛹阶段, 实现对特定细胞种群的长期成像和跟踪, 并与有效的 rna 干扰介导的基因失活相兼容。

视频: Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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, ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

果蝇蛹中炎症细胞动力学的长期体内追踪

摘要

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此视频中的章节

流式细胞仪分析小鼠主动脉内流的免疫细胞

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

长期慢性

跟踪小鼠骨髓单核细胞

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

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

股骨窗口商会模型

无创

从小鼠在原代肝细胞培养物的炎性反应的诱导

支气管肺泡灌洗小鼠肺分析炎症细胞浸润