我们感谢Christine Holt和Bill Harris在共聚焦显微镜方面的帮助。我们感谢Darren Gilmour的荧光计时器骨干结构，感谢Anna Huttenlocher的Tol2-Lyz骨干载体。我们感谢Steve Renshaw的Tg（mpx:GFP）i114 系列。这项工作得到了MRC（MR/L019523/1）、惠康信托基金会[204845/Z/16/Z]的支持。艾萨克·牛顿信托[12.21（a）i]和艾萨克·牛顿信托赠款19.23（n）。C.C.得到了MRC DTP学生奖学金的支持，部分得到了惠康信托基金会的支持[204845/Z/16/Z];艾萨克·牛顿信托 [12.21（a）i] 授予。H.W.得到了MRC DTP学生会的支持。H.P.得到了惠康信托博士奖学金（105391/Z/14/Z）的支持，部分得到了惠康信托[204845/Z/16/Z]的支持。Isaac Newton Trust [12.21（a）i] 授予和 MRC （MR/L019523/1）。

注意:所有斑马鱼均根据ARRIAGE指南和英国内政部法规，1986年英国动物（科学程序）法进行饲养。
1. 转基因报告基因斑马鱼幼虫的生成，用于白细胞受体运输成像
<ol><li>生成基于Tol2的构建体，用于感兴趣的荧光标记受体的组织特异性表达。对于中性粒细胞，使用来自溶菌酶C15 和髓过氧化物酶基因16的启动子序列。该构建体可以设计为与单个荧光蛋白（例如GFP），串联荧光计时器（例如，快速折叠的GFP和较慢成熟的tagRFP8，14，17）或报告基因GFP和对照膜标记物9 的双歧表达的融合（选择方法时的注意事项参见 讨论 ）。 注意:该构建物不概括内源性水平的受体表达，但有助于在感兴趣的细胞类型中获得高水平的受体表达。查阅有关类似受体3，5，6，8，9，10，14的文献以确定荧光标签的位置。</li>
	<li>按照标准饲养做法18建立一个包含野生型成年雄性和雌性的水箱，在产卵前一天用屏障隔开。</li>
	<li>在卵产卵当天，准备转基因混合物进行显微注射，其中含有12.5 ng / μL的Tol2 DNA构建体和17.5 ng / μL转座酶mRNA7。在早上灯光亮起后不久（不同鱼类设施可能会有所不同），从鱼缸中移除屏障，并在15分钟内收集卵以进行mRNA注射。 注意:确保DNA溶液不含RNase，以避免混合物中转座酶mRNA的降解。规避这种情况的一种选择是用单独的Tol2构建体和转座酶溶液注射卵子。</li>
	<li>遵循斑马鱼卵的转基因和显微注射的标准方案19。</li>
	<li>将1nL溶液注入单细胞阶段胚胎的细胞中。 注意:当注射细胞内并丢弃可能不在细胞内的注射时，表达结果更加一致。单细胞期胚胎是针对单细胞期胚胎的，因为在注射2-16个细胞期胚胎时，每个细胞的体积注射是可变的。一种选择是将单细胞阶段注射与稍后的注射分开，以防它们具有不同的功效。</li>
	<li>在当天晚些时候检查注射的胚胎，并取出未受精或死亡的卵子，以保持离合器健康。</li>
	<li>在受精后3天（dpf），在荧光显微镜下筛选幼虫。该标志物在嗜中性粒细胞中可见，特别是在富含这些细胞的尾部造血组织（CHT）中。 注意:标记的细胞的百分比因不同的结构而异，但通常预计20-60%的嗜中性粒细胞表达该结构。较低的百分比通常预示着成人阶段的筛查更多。在养鱼之前，用更高分辨率的成像方法，在这些胚胎的样本中验证受体在膜上的正确定位是一种很好的做法。</li>
	<li>按照标准饲养实践培养阳性幼虫20.这些代表F0一代。</li>
	<li>在3个月大时，为创始人筛选F0鱼。将个体鱼与非转基因野生型杂交，并通过在解剖范围下观察，以3 dpf筛选其后代以表达受体。根据转基因的成功，其因每个结构而异，在杂交的子集中观察到正后代的百分比。 注意:为了良好的转基因，大约三分之一到一半的成年人会产生积极的后代。离合器中阳性后代的百分比随插入的转基因的拷贝数而变化，并且可以在10-60%之间。跟踪离合器内的孟德尔比率以鉴定单次插入转基因是有帮助的（通过寻找50%的阳性幼虫比例，在F2离合器中更容易识别这些比率）20。</li>
	<li>培养代表F1一代的正后代。</li>
	<li>在3个月大时，以相同的方式筛选F1成人，以建立稳定的F2转基因系。</li>
	<li>在验证转基因的嗜中性粒细胞特异性表达后，对F2幼虫进行实验。 注意:在转基因过程中，人们可能会观察到不同水平的受体表达，建议保持不同的转基因离合器，以获得生物学问题最合适的表达水平。初始结果可在 F0 或 F1 幼虫中获得。</li>
</ol>2. 收集斑马鱼胚胎以评估白细胞伤口反应
<ol><li>在建立了稳定的转基因报告线后，在成年和雌性转基因鱼之间建立了杂交，并在第二天早上收集卵。</li>
	<li>在E3培养基（或卵水18）中在28.5°C下培养胚胎。</li>
	<li>任选地，在24 hpf下，将胚胎在含有50mL E3培养基的离心管中孵育5分钟。随后在E3培养基中冲洗3次，让管子静置几分钟，让胚胎沉降在底部，然后倾析并更换培养基。 注意:这为幼虫的感染暴露提供了一定程度的控制，这可能会影响白细胞在受伤期间的行为。</li>
	<li>漂白后，将胚胎保存在补充了0.003%的1-苯基-2-硫脲的E3培养基中，以防止黑色素合成。通常用于预防真菌感染的亚甲蓝不在这里添加，以尽量减少组织自发荧光。</li>
	<li>让幼虫自然孵化，并在嗜中性粒细胞丰富时以3 dpf使用21。</li>
</ol>3. 幼虫腹鳍伤
<ol><li>准备幼虫进行伤害。当观察到丰富的嗜中性粒细胞时，以2.5-3.5 dpf使用幼虫。将幼虫转移到补充有160-200mg / L三卡因MS222的E3培养基中。 注意:应提前制备三卡因的浓缩溶液并冷冻在等分试样中，并在当天解冻。</li>
	<li>将幼虫留在E3 + 三卡因培养基中15分钟，以确保它们被麻醉。用小画笔或类似工具轻轻触摸来检查他们的反应。</li>
	<li>在荧光解剖范围内选择具有转基因受体表达的幼虫。</li>
	<li>将幼虫转移到E3 +三卡因中的120毫米培养皿中进行伤害。使用无菌手术刀切除幼虫的腹鳍，同时在解剖范围内观察（图1）。 注意:我们的想法是进行足够深的切割以引起大量的嗜中性粒细胞增殖，但不切割CHT的血管。切口垂直于CHT轴，使得切口几乎到达CHT的血管。这需要一些练习，并且应该首先在监督下对不是为此目的产生的幼虫（例如，来自另一个实验的过量幼虫）进行。</li>
</ol>4. 准备幼虫进行活体成像
<ol><li>通过加热将低熔点（LMP）琼脂糖溶解在E3介质中，得到液体2%琼脂糖溶液。</li>
	<li>让该溶液冷却至60°C。 注意:将装有琼脂糖的烧瓶或管保存在60°C的培养箱中，以避免不同幼虫的安装之间的琼脂糖设置。</li>
	<li>移液0.5毫升液体LMP + E3在玻璃底培养皿中用于显微镜成像。</li>
	<li>将受伤的麻醉斑马鱼幼虫与0.5毫升E3 + 2x三卡因一起移液到玻璃底培养皿中。</li>
	<li>轻轻混合两种溶液，得到1%LMP / 1x三卡因琼脂糖/ E3溶液，避免产生气泡。将胚胎定向并轻轻向下推，使鱼的尾部尽可能靠近玻璃。 注意:使用倒置显微镜成像时，要成像的组织必须尽可能靠近玻璃底部。幼虫的方向设置必须快速，以便在幼虫定位之前不会设置琼脂糖。</li>
	<li>让琼脂糖溶液冷却并凝固5-10分钟。通过用小油漆刷或笔尖轻轻触摸琼脂糖凝胶来测试琼脂糖是否凝固。</li>
	<li>琼脂糖为固体后，向成像平板中加入2 mL E3，并补充0.2mg / mL三卡因。</li>
</ol>5. 实时共聚焦成像
注意:在旋转盘共聚焦显微镜或等效物上对胚胎进行成像（图2）。也可以使用激光扫描显微镜，但动力学的时间分辨率将更具限制性。在携带受伤的幼虫之前准备成像设置，以便在受伤后尽快成像反应。中性粒细胞在5分钟内到达伤口，第一个到达细胞中的受体可能在此时间范围内内化。通过实践，可以在受伤后15分钟进行成像。
<ol><li>按照制造商的说明打开显微镜:激光，相机和计算机。</li>
	<li>使用采集软件设置成像设置。根据先前的实验选择合适的荧光团激光器和近似曝光时间（用488nm获取GFP，用561nm激光获取tagRFP）。</li>
	<li>琼脂糖凝固后尽快将装有安装的胚胎的板转移到共聚焦成像旋转盘平台上。</li>
	<li>使用显微镜目镜通过舞台操纵杆找到盘子里的鱼。</li>
	<li>使用对焦旋钮对焦伤口区域。 注意:要找到感兴趣的区域，使用低倍率空气物镜（10x）可能更容易。</li>
	<li>选择要在伤口周围成像的字段。使用具有高数值孔径的30x/40x物镜以获得足够的分辨率。</li>
	<li>使用软件按钮调整曝光时间，以便以良好的对比度看到荧光标记物，但不会使信号饱和。 注意:曝光时间必须尽可能短，以最大限度地减少荧光曝光，并在延时中最大化时间分辨率。激光功率取决于激光器的状况，但需要调整到允许足够低的曝光时间以进行动态成像的水平。</li>
	<li>使用软件按钮选择要映像为 z 堆栈的卷</li>
	<li>在所需的时间内每30秒设置一次时间流逝。 注意:对于无菌腹鳍伤口，最大募集在2-3小时观察到。</li>
	<li>在启动延时摄影之前，请拍摄明场快照以记录视野。如果可能，在延时短片中获取明场。</li>
</ol>6. 斑马鱼中性粒细胞受体内化的定量
<ol><li>记录图像采集的时间间隔和图像的像素大小。记录成像开始后受伤后的分钟数。</li>
	<li>通过将图像拖动到软件界面上，使用斐济打开图像数据集，使用时间滑块为每个数据集选择一个具有代表性的感兴趣帧，例如，在缠绕后1-1.5小时，然后保存。</li>
	<li>继续使用 MATLAB 处理图像数据集。</li>
	<li>创建一个新脚本，并包括用于图像读取的函数（在 补充文件 1 中称为"select_neutrophils.m"的脚本中称为"select_neutrophils.m"的脚本中，用于质心定义）、打开（ 补充文件 1 中的第 11 行）和手动选择图像上的点（ 补充文件 1 中的第 12 行）。</li>
	<li>通过运行此脚本（补充文件1）打开感兴趣的框架，通过目视检查识别要分析的嗜中性粒细胞，单击它们并记录腹鳍伤口和CHT中其质心的估计。 注意:CHT中非动员的嗜中性粒细胞可作为受体分布保持不变的嗜中性粒细胞的内部参考。这允许将伤口处细胞的对比度值归一化到内部参考。</li>
	<li>使用活性轮廓技术22 继续分割每帧中的嗜中性粒细胞，如以下步骤所述。</li>
	<li>创建一个函数以包含按活动等值线进行分割所需的元数据（即迭代次数、等值线偏差、估计质心等）。22 （见 补充文件2中的"伤口数据.m"）。</li>
	<li>创建一个调用函数"wound_data.m"的脚本，以输入分割每个嗜中性粒细胞所需的信息（ 补充文件 3 中脚本中称为"calc_contrast.m"的第 28 行）。</li>
	<li>包含在用于图像读取的脚本命令中（ 补充文件 3 中的第 32 行）。</li>
	<li>添加与输入图像大小相等的黑色图像（即像素值为零的图像）的生成（ 补充文件3中的第44行），并定义每个中性粒细胞质心周围10×10像素的正方形（ 补充文件3中的第45行）。</li>
	<li>包括使用活动轮廓的中性粒细胞分割（补充文件3中的第48-49行）和去除小的假检测物体（ 补充文件3中的第52行）。 注意:初始分割等值线是质心周围的正方形，它通过基于像素强度、迭代次数和轮廓偏差的主动轮廓技术演变而来。分割的结果是一个二进制掩码，其中所有像素的值为 0，而中性粒细胞区域的像素值为 1。</li>
	<li>包括分割的二进制图像与原始图像的乘法，仅获得中性粒细胞的像素强度，图像的其余部分不是数字，因此它不有助于计算（ 补充文件3中的第56-57行）。</li>
	<li>添加每个中性粒细胞 （GLCM）14，23 的灰度共现基质的计算（ 补充文件 3 中的第 61 行）。GLCM是图像的另一种表示形式，显示像素在像素强度方面的相对位置。</li>
	<li>包括基于GLCM的嗜中性粒细胞造影剂的计算（ 补充文件3中的第62，65行）。对比度指标测量相邻像素之间的强度差异。将像素与相距一定距离的像素进行比较，可以根据强度局部峰值的大小进行经验调整。作为指示，对于像素尺寸为0.389μm的图像，当受体显示囊泡分布时，每个亮点都在5像素的范围内。因此，以间隔5个像素的像素比较强度。</li>
	<li>添加命令以单独保存腹鳍中单个嗜中性粒细胞的值（ 补充文件 3 中的第 68 行）。</li>
	<li>包括所有CHT嗜中性粒细胞的平均中性粒细胞对比度值的计算，其方式与伤口处的嗜中性粒细胞相同（补充文件3中的第72-119行）。对于CHT嗜中性粒细胞，将函数称为"cht_data.m"（ 补充文件4中的第77行）。</li>
	<li>包括伤口处单个嗜中性粒细胞的对比度值与上述步骤6.16中计算的CHT嗜中性粒细胞的平均对比度的归一化（即除法）（ 补充文件3中的第122行）。这给出了一个标准化的对比，反映了相对于对照无反应细胞，受体在单个反应细胞中的外观是多么"点"（图3 和 图4）。</li>
	<li>通过单击软件中的运行符号来运行脚本（补充文件 3）。</li>
	<li>针对不同情况重复所有步骤。</li>
	<li>使用统计软件（见 材料表）通过创建列表来导入不同条件的结果，绘制结果并进行统计检验以检查平均值之间差异的显著性。 注意:分析代码也可以在GitHub的 https://github.com/LeukocyteMotionAndDynamics/ReceptorTraffic</li>
</ol>7. 早期胚胎中的趋化因子反应测定
注意:这是一个可选的副实验，允许测试响应候选趋化因子的受体分布变化，并且独立于上述关于受体构建体的中性粒细胞表达的实验。使用该技术很难确定配体诱导的受体之间运输的差异，因为配体水平饱和14。然而，如果人们看到该系统中受体的配体内化，这可能表明在配体身份不明确的情况下，配体被受体识别。这是有用的，因为在已建立的细胞系（如HEK293T细胞14 ）中表达趋化因子受体可能很麻烦。
<ol><li>设置野生鱼类（例如AB）的杂交，并在抬起分离器后不久的第二天早上收集卵（如上所述）。</li>
	<li>注射 100 pg 荧光标记的受体 mRNA（例如 Cxcr1-FT），以及 100 pg 的 mRNA 用于膜标记（例如，膜 CFP）。在混合物中包括不同剂量的mRNA用于趋化因子配体。 注意:作为指示，150 pg Cxcl8a mRNA给予荧光Cxcr1-FT的显着内化（见 图5）。</li>
	<li>用E3培养基冲洗胚胎并在28°C下孵育。</li>
	<li>在约7 hpf下，在荧光解剖仪上测试mRNA的表达，并选择胚胎进行成像。</li>
	<li>提前准备0.8%LMP琼脂糖，并在60°C的热块中保存在玻璃管中。使用玻璃移液器操纵胚胎。使用每只手中的一对镊子轻轻地去皮化胚胎。</li>
	<li>用玻璃移液器吸出单个去壳胚胎，确保尖端没有气泡。轻轻地将胚胎释放到琼脂糖管中，使其沉入管中。</li>
	<li>从琼脂糖管中吸出胚胎，沿途收集一些液体琼脂糖。轻轻地将胚胎释放到玻璃底成像皿的中心。快速旋转胚胎，使动物杆朝向培养皿的底部（使用倒置显微镜时，这一侧必须最接近物镜）。 注意:当琼脂糖仍在凝固时，可能需要重新调整胚胎的方向。</li>
	<li>琼脂糖凝固后，补充2毫升E3培养基。 注意:与幼虫相比，这个阶段的胚胎非常脆弱，需要一些练习来去皮化和安装。重要的是要尽可能轻柔地抽吸和释放，以避免胚胎破裂。</li>
	<li>重复该过程，目标是每培养皿加载3-5个胚胎。</li>
	<li>在倒置共聚焦显微镜上对胚胎进行成像（参见 材料表）。使用40x/1.3 NA油物镜获得足够高的分辨率。在示波器上分别显示 405、488 和 552 nm 的 mCFP、sfGFP 和 tagRFP。调整滤镜和设置以具有高对比度，同时避免饱和并最大限度地减少通道之间的泄漏。</li>
	<li>针对不同情况重复安装和成像。</li>
</ol>

<ol>
	<li>Rot, A., von Andrian, U. 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作者声明没有利益冲突

所描述的方法允许在对组织损伤的炎症反应期间原位响应于内源性配体的受体动力学的实时成像。Cxcr1/ Cxcr2嗜中性粒细胞报告基因的使用可以扩展到其他生理环境，例如感染，肿瘤模型或其他类型的组织损伤14，25，26，27。此外，转基因抢救系，其中内源性受体被外源性突变受体抑制和挽救，可以为剖析特定中性粒细胞迁移模式在免疫反应中的重要性提供有用的工具。例如，具有脱敏受损的Cxcr1受体突变体在炎症部位14引起更突出的中性粒细胞聚集。这种功能表型的增益可用于了解中性粒细胞聚集在不同生理过程中的作用，例如伤口修复，传染病或肿瘤进化，以及补充受体敲低/敲除实验。该方法还为扩大现有报告员的范围提供了基础。荧光报告基因的选择很重要，取决于生物学问题。我们发现，与上皮细胞相比，中性粒细胞中这些趋化因子受体的组成性周转率很高，并且具有快速成熟（例如sfGFP）的报告基因需要在稳态下报告膜水平并解析中性粒细胞刺激时的差异8，14。因此，sfGFP / tagRFP的膜比不适用于测量配体诱导的这种细胞类型的内化，但tagRFP的模式允许跟踪受体的细胞内命运，这可能在某些研究中有用。我们还发现，tagRFP更集中的细胞内信号对于筛选单个幼虫是有用的。测量质膜上受体水平的另一种方法是在同一转基因9 或独立转基因14中的嗜中性粒细胞中共表达荧光膜标记物。在前一种情况下，转基因将为筛选鱼类提供额外的手段，并且标记物和受体之间的表达水平相当。后一种方法将更加模块化，因为带有受体报告器的斑马鱼线可以与不同的报告线组合。在任何一种情况下，值得注意的是，在聚集性中性粒细胞中，受体水平的膜定量具有挑战性（见下文）。最后，我们注意到该协议的可能扩展是通过免疫组织化学跟踪实时成像，以进行更详细的定位分析。
Tol2转基因系统已建立7 ，溶菌酶C启动子已被广泛用于中性粒细胞表达11，15。因此，转基因方法相对简单，并且使用该启动子达到的表达水平足够高，可以为分析受体动力学提供足够的对比度。一个可能的局限性是表达水平不概括内源性受体表达水平。可以部署新的CRISPR技术来为特别感兴趣的受体建立敲入线28。这些技术仍然很麻烦，可能无法保证亚细胞成像所需的表达水平，但它们的成功开发将是理解内源性信号传导动力学的重要突破。功能验证对于用转基因受体构建体解释数据非常重要。例如，配体识别测定可用于确定荧光融合蛋白具有功能性，并且可用于拯救敲除表型以确定转基因嗜中性粒细胞表达水平与功能14相容。最后，验证受体融合的更直接方法是利用体外功能测定，将标记的受体与未标记的版本14一起使用。
定量方法解决了体内嗜中性粒细胞准确膜分割的特定困难。在上皮性质的细胞中，可以通过将膜受体水平归一化为对照标记物来自动执行受体水平的定量，其可以串联或单独表达9。事实上，我们已经应用了这种方法，当在胃压胚胎中使用配体识别测定14时。然而，嗜中性粒细胞在体内经历复杂，快速的细胞形状变化，使得在2D和3D14中膜分割困难。当嗜中性粒细胞聚集时，这更具挑战性，这发生在许多生理环境中29。造影剂克服了这一限制，因为它不需要膜分割，而是反映了细胞中受体分布的整体状态（膜性/光滑性与囊泡性/点状）。重要的是要注意，对比度指标可能会受到图像整体对比度的影响，因此需要将单个细胞值归一化为内部参考，以考虑不同胚胎/样品中信号的变异性。例如，我们使用了CHT中无反应性嗜中性粒细胞的平均细胞对比度值（即，保持静止且不会迁移到腹鳍的嗜中性粒细胞）14。另一种可能性是使用同一单元格中对照标记的对比度值进行归一化。当无反应细胞的内部参考不可用时，这将提供一种解决方案，并且可能解决不同条件之间受体动力学的更精细的定量差异。
影像学检查的位置是另一个需要考虑的变量。这里选择腹鳍伤口的原因，而不是更常用的尾鳍伤口模型16，30，是因为伤口部位靠近嗜中性粒细胞居住/迁移起源的部位。这加快了测定的时间线，因为中性粒细胞到达所需的时间相对较短。此外，它还提供了在迁移源（CHT）和目标位置（伤口）捕获细胞行为的机会。这在这里是相关的，因为亚细胞成像所需的空间和时间分辨率很难与大视场或多位置扫描相结合。因此，腹鳍伤口测定允许跟踪迁移反应从迁移起源的进化，并同时捕获无反应细胞中的非特异性受体波动。如上所述，后者对于量化目的很有用，因为它为非特异性动力学提供了内部参考。在其他系统中，可能不可能有这样的内部参考，在这种情况下，共表达膜标记物的对比度值将提供替代对照。
总之，我们预计该方法适用于其他系统，并且可以用于各种目的。例如，相同的报告基因可用于其他炎症环境，如感染环境或其他疾病模型。斑马鱼受体报告基因系的库可以扩展到其他信号传导受体，以了解信号传导机制或报告体内配体动力学。该方法可以与敲低/敲除技术相结合，以询问观察到的动力学的机制基础。例如，配体表达的扰动可以指示配体对观察到的受体动力学的依赖性。未来，我们设想可以进一步完善该系统，以纳入记者的敲入插入。最终，使用这种方法的研究结果将为斑马鱼群落之外提供有价值的新见解，因为哺乳动物中这些信号受体的保护以及在大型生物体中进行这些研究的相对挑战。

白细胞迁移对免疫反应至关重要。免疫细胞是典型的迁移细胞，它们非常能够穿越组织和血管，并感知一系列化学引导线索，以定向地向微生物或其他重要的宿主细胞迁移。正确的指导依赖于对化学牵连剂的识别，其中趋化因子代表了最突出的类别1。趋化因子由高度特异性的七跨膜G蛋白偶联受体识别。在趋化因子结合后，趋化因子受体改变构象，导致相关的三聚体G蛋白的活化及其解离成功能信号子亚基，促进细胞骨架变化和定向迁移1。其次，趋化因子受体被磷酸化，并且这种修饰导致对引诱剂的脱敏，随后可以从细胞表面2快速再敏化/再循环或细胞内降解和下调。这些受体动力学影响细胞接收的信号传导的持续时间和剂量，但它们如何影响白细胞迁移行为在体内很难阐明。
在传统哺乳动物系统中跟踪活白细胞中的受体动力学面临几个挑战。对于现场研究，受体与荧光蛋白的融合必须在细胞中表达。这在原代白细胞中具有挑战性，特别是在嗜中性粒细胞中，迄今为止的研究已经使用替代中性粒细胞系来表达趋化因子受体3，4。生成转基因小鼠模型，其中白细胞表达荧光受体或具有信息性贩运缺陷的突变受体5，6，需要投入大量的时间和资源。即使在这些情况下，活体动物中成像受体动力学的成像分辨率和对比度也可能受到限制，并且研究已经在固定组织切片中使用了免疫组化学5。鉴于这些技术挑战，我们对化学吸引受体动力学如何影响活组织环境中的细胞行为的理解目前有限。
在这里，我们提供了一种监测斑马鱼中性粒细胞受体运输的方法。斑马鱼在遗传上是可处理的，像老鼠一样，但通过使用有效的转座子系统和直接的受精卵操作7，转基因相对更直接。透明幼虫非常适合成像。趋化因子受体动力学已通过与荧光报告基因8，9，10的相应融合表达在原始生殖细胞和侧线原始中可视化。斑马鱼幼虫具有成熟的嗜中性粒细胞，与哺乳动物中性粒细胞相比具有高度保守的遗传和细胞特性。亚细胞信号动力学，如细胞骨架动力学和极性调节因子，已经通过产生相应的转基因品系11，12，13在这些细胞中可视化。最近，我们可视化并功能分析了中性粒细胞在组织损伤炎症反应过程中的趋化因子受体动力学14。在这里，我们描述了嗜中性粒细胞趋化因子信号传导的转基因报告线的产生，用于实时成像的胚胎制备，用于研究嗜中性粒细胞信号传导的伤口测定以及用于获取和分析图像的方案。我们还提供了一种侧方案来测试趋化因子受体对候选配体的反应，这在尝试在未表征的受体中建立配体识别模式时很有用。这些技术可以与进一步的遗传操作结合使用，例如抑制内源性趋化因子表达或产生具有改变配体诱导的运输的突变受体，以询问特异性信号动力学如何影响体内白细胞行为。表达荧光标记的趋化因子受体的转基因品系也可以用作内源性趋化因子梯度的报告基因，否则很难通过直接抗体染色检测到。所描述的方法为将报告基因的产生扩展到其他免疫信号传导受体提供了空间。

<table>
	<tbody>
		<tr>
			<td>1-phenyl-2-thiourea</td>
			<td>Sigma-Aldrich</td>
			<td>P7629-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL CellStar Centrifuge Tube</td>
			<td>Grenier Bio-One Limited</td>
			<td>210270</td>
			<td></td>
		</tr>
		<tr>
			<td>Clark capillary glass</td>
			<td>Harvard Apparatus</td>
			<td>30-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleanline Thin Bleach</td>
			<td>Scientific Laboratory Supplies</td>
			<td>CLE0301</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scope</td>
			<td>Nikon</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>Dri Block heater</td>
			<td>Techne</td>
			<td>FDB02AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>National Institutes of Health, Bethesda, MD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Jewelers Dumont No. 5</td>
			<td>Sigma-Aldrich</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom microwell dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pasteur pipette</td>
			<td>Fisherbrand</td>
			<td>11795098</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Ambion mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>10391175</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The MathWorks, Inc., Natick, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M-2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection system</td>
			<td>Parker</td>
			<td>Picospritzer III</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>Flaming/Brown P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo-Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PerkinElmer Spinning Disk UltraVIEW ERS, Olympus IX81 spinnng disk confocal microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (120mm)</td>
			<td>Grenier Bio-One Limited</td>
			<td>632180</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tailing Kit</td>
			<td>Invitrogen</td>
			<td>AM1350</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small paintbrush</td>
			<td>N/A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral Applied Research LMM5 laser module</td>
			<td></td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scalpel Blade No. 23</td>
			<td>Swann-Morton</td>
			<td>233-5528</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine MS222</td>
			<td>Sigma-Aldrich</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Volocity software</td>
			<td></td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Yokogawa CSU10 spinning disc confocal scanner</td>
			<td>Yokogawa</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of chemokine receptors in zebrafish neutrophils during wound responses

通过化学梯度引导白细胞对于免疫反应至关重要。中性粒细胞是第一批被招募到组织损伤部位的细胞，在那里它们执行关键的抗菌功能。它们被贩运到这些位点是由几种炎症性化学诱捕剂（包括趋化因子）精心策划的。在分子水平上，化学吸引剂信号传导受相应受体的细胞内运输的调节。然而，目前尚不清楚趋化因子受体的亚细胞变化如何影响细胞和组织水平的白细胞迁移动力学。在这里，我们描述了一种在组织损伤的炎症反应期间，中性粒细胞趋化因子受体动力学的实时成像和定量分析的方法。这些工具表明，斑马鱼中性粒细胞中差异化因子受体的运转协调中性粒细胞聚集和在组织损伤部位的扩散。这对我们理解炎症反应如何自我解决具有重要意义。所描述的工具可用于了解各种生理和病理环境中的嗜中性粒细胞迁移模式，并且该方法可以扩展到其他信号传导受体。

腹鳍损伤后，在30-60分钟内从CHT快速中性粒细胞动员到腹鳍并在伤口边缘聚集（图1）。我们使用旋转盘共聚焦显微镜可视化两种趋化因子受体Cxcr1和Cxcr2的分布，它们由斑马鱼嗜中性粒细胞24 表达并识别Cxcl8a和Cxcl8b14。我们生成了两个相应的转基因品系，Tg（lyz:Cxcr1-FT）和Tg（lyz:Cxcr2-FT），其中嗜中性粒细胞表达受体的荧光计时器（FT）构建体，即与sfGFP和tagRFP的串联融合（图2 和参考文献14）。使用这两种荧光团的目的是允许监测广泛的受体命运，并提供质膜上蛋白质周转时间的估计，因为新合成的受体会在8，14岁时发出绿色荧光并逐渐变红。然而，发现这些受体在中性粒细胞质膜处具有快速的组成性周转，并且停留时间短于tagRFP的成熟时间，sfGFP显示膜定位，tagRFP显示稳定状态下的囊泡定位（补充视频1 和参考文献14）。因此，我们专注于sfGFP的分布，以监测配体诱导的组织损伤部位的内化。使用对比度指标量化受体分布的模式，该指标报告了相邻像素之间的强度差异。其基本原理是，当受体分布膜状且平滑时，对比度值较低。当受体分布是水疱状的并且更点状时，则对比度值高（图3）。
另一种方法是量化受体水平（sfGFP强度）与对照膜标志物水平（例如膜CFP（mCFP））水平的比率（图3）。这两种方法都可以检测受体内化，如细胞中更多的囊泡受体分布模式（较高的对比度值）或膜处较低的受体水平（较低的sfGFP / mCFP比率）所示。然而，造影剂指标还可以检测伤口处嗜中性粒细胞簇中的受体内化，其中膜分割不太准确且不适用（图3）。使用这个指标，我们能够量化伤口处中性粒细胞中Cxcr1和Cxcr2之间明显的差异（图4 和 补充视频2）。Cxcr1-FT在位于伤口的细胞中内化，而Cxcr2-FT在伤口的嗜中性粒细胞中保持膜状（图4A-C， 补充视频2 和 补充视频3）。通过吗啉诺处理抑制Cxcl8a和Cxcl8b，在伤口处差异化影响Cxcr1-FT内化（图4C，D）。为了进一步验证Cxcr1-FT对Cxcl8a的反应，我们在早期胚胎中进行了趋化因子反应测定。我们发现Cxcr1-FT在Cxcl8a共表达的胚胎中显着内化（图5）。总而言之，这些结果表明，所描述的方法可用于测量嗜中性粒细胞中趋化因子诱导的受体内化，并建立介导这些作用的配体的身份。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61679/61679fig01.jpg"> 图1:中性粒细胞向腹鳍伤口的迁移。 （A）（左）3 dpf幼虫的卡通，显示了尾部造血组织（CHT），金星循环（VC，蓝色），腹鳍（VF）和伤口部位的位置。（右）卡通描绘了伤口区域（W），中性粒细胞从CHT中动员起来并在伤口处聚集。CHT组织的尾静脉丛（CVP）以蓝色绘制。（B）明场图像（左）和共聚焦投影（右）显示Tg（mpx:GFP）幼虫在伤后2 h腹鳍伤口和嗜中性粒细胞的分布。虚线显示 VF 和 CHT 轮廓。比例尺 = 25 μm。根据参考文献14 （http://creativecommons.org/licenses/by/4.0/）修改的卡通和荧光图像。 <a href="https://www.jove.com/files/ftp_upload/61679/61679fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61679/61679fig02.jpg"> 图2:嗜中性粒细胞中趋化因子受体运输的实时成像。 （A）用于中性粒细胞特异性转基因表达Cxcr1-FT（荧光计时器）和Cxcr2-FT的构造.中性粒细胞在3 dpf转基因幼虫（Tg（lyz:Cxcr1-FT）头部的共聚焦投影，顶部;Tg（lyz:Cxcr2-FT），底部）显示tRFP（洋红色）和sfGFP（绿色）通道。比例尺 = 20 μm.（B） 3 dpf 幼虫的解剖方案如图 1A 所示。幼虫下方是描绘伤口区域（W）的方案，其中嗜中性粒细胞从CHT（顶部）动员或在进入腹鳍（底部）时进行趋化性。虚线正方形表示右侧快照中成像的区域。（C）Tg（lyz:Cxcr1-FT）幼虫（显示sfGFP）中的嗜中性粒细胞，从CHT（顶部面板）动员或趋化性向伤口（底部面板）。箭头显示随时间变化的相同单元格。右图上的时间点是左图之后经过的分钟数。比例尺 = 10 μm. （D） 化学因子受体运载活体成像实验方法的示意图。根据参考文献14 （http://creativecommons.org/licenses/by/4.0/）修改的面板A-C。 <a href="https://www.jove.com/files/ftp_upload/61679/61679fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61679/61679fig03.jpg"> 图3:受体动力学的定量示例。 对伤口处的单粒细胞（蓝色）或簇状嗜中性粒细胞（绿色）或CHT中的非活动性中性粒细胞（红色，橙色）进行分段，并用不同的方法进行分析以比较结果。使用两种方法分析显示的相同示例单元格，以将图像中看到的内容与提取的值范围相关联。（A） 根据 sfGFP 通道中的轮廓定义对所选示例像元的表面进行分割。（B）根据CFP通道中的轮廓定义对所选示例细胞的膜进行分割。随后对sfGFP /CFP进行了比例分析。（D）在C所示的示例单元格上计算sfGFP / CFP的比率.误差线表示来自单个单元格的S.E.M.，在n>1的情况下，此处的值不用于统计分析，而仅用于举例说明使用不同定量方法获得的测量结果。比例尺 = 10 μm。图修改自参考文献14（http://creativecommons.org/licenses/by/4.0/）。<a href="https://www.jove.com/files/ftp_upload/61679/61679fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61679/61679fig04v2.jpg"> 图 4:Cxcr1 和 Cxcr2 对伤口的响应差异动力学。 （A）在伤口后80分钟（显示sfGFP通道）时Tg（lyz:Cxcr1-FT）或Tg（lyz:Cxcr2-FT）幼虫中嗜中性粒细胞在伤口上的共聚焦投影。比例尺 = 10 μm。 （B）伤口处的Cxcr1-FT中性粒细胞（左）和Cxcr2-FT（右）放大。绿色受体以灰色显示。（C）归一化对比（每个个体中性粒细胞的对比度归一化为CHT中非动员细胞的平均对比度）。cxcl8a 是指注射剪接阻断剂以及用于 cxcl8a 的翻译阻断吗啉。cxcl8b 是指用剪接阻断吗啉诺注射 cxcl8b。对于Tg（lyz:Cxcr1-FT）:n = 24个细胞（CHT），n = 来自8个幼虫的47个细胞（伤口）。对于具有吗啉的Tg（lyz:Cxcr1-FT）:来自5个幼虫的n = 28个细胞（Cxcl8a-MO），来自5个幼虫的n = 16个细胞（Cxcl8b-MO）。对于Tg（lyz:Cxcr2-FT）:来自3个幼虫的n = 10个细胞（CHT）和n = 20个细胞（伤口）。从1-5次成像过程中获得的独立幼虫收集数据。Kruskal-Wallis测试与Tg（lyz:Cxcr1-FT）的Dunn多重比较测试，Tg（lyz:Cxcr2-FT）的双尾未成对曼-惠特尼测试。（D）用cxcl8a吗啉（MO）和cxcl8b MO（右）处理的转基因幼虫Tg（lyz:Cxcr1-FT）中嗜中性粒细胞的共聚焦投影，对鳍状伤口有反应（sfGFP通道以绿色显示）。在等效中性粒细胞积累的时间点拍摄的快照（左图中为伤口后85分钟，右图为伤口后45分钟）。比例尺 = 10 μm。图修改自参考文献14（http://creativecommons.org/licenses/by/4.0/）。<a href="https://www.jove.com/files/ftp_upload/61679/61679fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61679/61679fig05.jpg"> 图5:早期胚胎中的趋化因子反应测定。 将100 pg Cxcr1-FT mRNA注射到具有或不具有150 pg Cxcl8a mRNA的细胞阶段卵中，将100 pg的气体胚胎共聚焦切片显示Cxcr1-FT的表达和分布。绿色和红色受体显示在不同的通道中。控制膜CFP标记物（mCFP）显示在青色通道中。比例尺 = 20 μm。图修改自参考文献14 （http://creativecommons.org/licenses/by/4.0/）。 <a href="https://www.jove.com/files/ftp_upload/61679/61679fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
补充电影 1: Tg（lyz:Cxcr1-FT）（左）和Tg（lyz:Cxcr2-FT）（右）幼虫头部的转基因嗜中性粒细胞，3 dpf。sfGFP（绿色）， tagRFP （洋红色）.帧间隔为 30 秒，帧速率为 5 fps。比例尺 = 20 μm。视频源自参考文献14 （http://creativecommons.org/licenses/by/4.0/）。 <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie1.avi" target="_blank">请点击此处下载此视频。</a>
补充电影2: Tg（lyz:Cxcr1-FT）（左）和Tg（lyz:Cxcr2-FT）（右）转基因幼虫中的中性粒细胞对鳍伤有反应。影片在上线后 10 分钟内开始，持续 60 分钟 sfGFP（绿色）、tagRFP（洋红色）。帧间隔为 30 秒，帧速率为 10 fps。CHT = 尾部造血组织。VF = 腹鳍。比例尺 = 25 μm。视频源自参考文献14 （http://creativecommons.org/licenses/by/4.0/）。 <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie2.avi" target="_blank">请点击此处下载此视频。</a>
补充电影 3. 来自受伤的Tg（lyz:Cxcr1-FT）转基因幼虫（与视频2所示不同的幼虫）的嗜中性粒细胞的其他例子，以更高的分辨率获得，在CHT中或进入时显示受体内化（以绿色显示的sfGFP通道）和腹鳍趋化性。帧间隔为 30 秒，帧速率为 2 fps。比例尺 = 10 μm。视频源自参考文献14 （http://creativecommons.org/licenses/by/4.0/）。 <a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary_Movie3.avi" target="_blank">请点击此处下载此视频。</a>
补充文件1:<a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%201.m" target="_blank">请点击这里下载此文件。</a>
补充文件2:<a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%202.m" target="_blank">请点击这里下载此文件。</a>
补充文件3:<a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%203.m" target="_blank">请点击这里下载此文件。</a>
补充文件4:<a href="https://cloudflare.jove.com/files/ftp_upload/61679/Supplementary%20file%204.m" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses at JoVE.com

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

在这里，我们描述了对斑马鱼嗜中性粒细胞的化学引诱剂受体动力学进行实时成像和定量分析的方案。

在伤口反应期间斑马鱼中性粒细胞中趋化因子受体的实时成像

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

live-imaging-chemokine-receptors-zebrafish-neutrophils-during-wound

Research

JoVE Journal

Immunology and Infection

2.4K 次观看.  University of Cambridge. 在这里，我们描述了对斑马鱼嗜中性粒细胞的化学引诱剂受体动力学进行实时成像和定量分析的方案。

视频: Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.