The authors have no acknowledgements.

所有的动物实验都按照健康指导全国学院进行，由托莱多大学的机构动物护理和使用委员会批准。 1. pIL1-红色荧光蛋白的小鼠的表型注:后代是通过育种杂pIL1-DsRed的小鼠与野生型（WT）C57BL6小鼠产生。三，四周龄的幼崽被认为是准备表型。小鼠颌下腺出血如下稍作修改23既定的协议。 <ol><li> 从乳鼠的全血白细胞分离 <ol><li>加入20μl肝素的每个1.5 ml离心管。 </li><li>获得从笼子1小狗。记录小狗的识别标签。它不需要从颌下静脉麻醉采血前幼仔。 </li><li>颈部颈背举行的小狗和皮尔斯在次颌下静脉使用5mm柳叶刀ê颊囊。适当用力创建一个小的穿刺，让血滴从切入点散发。使用新的针头为每小狗。 </li><li>收集3 - 每个小狗血5滴在含有肝素的离心管中。清洁穿刺部位和施加压力，以促进止血。 </li><li>放小狗在新笼子和笼返回到动物设施之前观察30分钟。 </li><li>从已知的表型作为阳性和阴性对照的成年小鼠收集血液。 </li><li>加入500微升的红血细胞裂解缓冲液中向每个管中，涡流管，并孵育5 - 在冰上10分钟。 </li><li>慢慢加入400 - 500微升的胎牛血清（FBS），在每个管中的细胞悬浮液的下方。一个明显的界面可以在上层的细胞悬浮液和FBS的下层之间被观察到。 </li><li>帽管和离心样品在1500×g下5分钟。 </li><li>重复步骤1.1.7 - 1.1.9皮草疗法去除红细胞。如果裂解足以在第一时间不要重复。粒料应该很难离心后辨别。 </li></ol></li><li> 脂多糖（LPS）刺激与文化 <ol><li>悬浮细胞在200微升完全罗斯威尔公园纪念研究所（RPMI）1640培养基。 </li><li>细胞悬液转移至血细胞计数管的流动。 </li><li>使脂多糖通过用990微升的完全RPMI 1640培养基中的混合10微升的LPS原液（1毫克/毫升）的工作溶液。 </li><li>加入20μl的工作溶液到200μl的细胞悬浮液的10微克/毫升的LPS。 </li><li>帽管松散孵育样品在37℃，5％的CO 2 4小时。 </li></ol></li><li> 流式细胞分析 <ol><li>打开流式细胞仪数据采集程序。设置为样本采集前采集模板。 </li><li>单击工具调色板上的点图工具。绘制FOrward散射（FSC）与侧散射（SSC）的情节。设置FSC和SSC电压参数的线性度。 </li><li>选择的FSC和SSC的通过流式细胞仪所允许的最低阈值。分别适用阈值200和50 FSC和SSC。 </li><li>内的FSC VS SSC情节，画出一个门G1，其包括单核细胞，粒细胞，和树突细胞，以及不包括碎屑，红血细胞和淋巴细胞。 </li><li>单击工具选项板直方图工具。绘制FL2（红色荧光通道）的直方图。使用阴性对照样品设置FL2信号的基线和阳性对照样品来绘制门包括所有的红色荧光蛋白阳性事件。 </li><li>在流式细胞仪的射流控制面板，设置在流体模式到"运行"和流速为"HI"（约60微升/分钟）。获得每个样品10000个事件。 </li><li>通过从G1栅极测量DsRed的阳性细胞的百分比确定小狗表型。 </li></ol></li></ol><p类="jove_title"&gt; 2。在pIL1-红色荧光蛋白的小鼠皮肤炎症的诱导<ol><li>麻醉由含有100毫克/千克氯胺酮，10mg / kg的甲苯噻嗪，和1毫克/千克乙酰丙嗪麻醉鸡尾酒的腹膜内（IP）注射小鼠。充分麻醉小鼠没有表现出后肢撤回到脚趾捏。 </li><li>申请脱毛霜鼠标两耳的背侧面。等待30秒到1分钟。擦拭耳表面用水润湿的棉花和允许空气干燥。 </li><li>使用千分尺测量耳的厚度，并在一个单独的笼子代替鼠标。观察到从麻醉恢复（〜15 - 30分钟）。要解决的脱毛产品引起的皮肤炎症，让前进一步的实验进行鼠标休息3天。 </li><li>由16.7毫克OX溶解于1ml丙酮中，混合750微升的OX /丙酮溶液与250微升的橄榄油制备1.25％（重量/体积）OX。通过混合750微升丙酮与250制备载体溶液＃181;橄榄油中的L. </li><li>由麻醉鸡尾酒（2.1）的腹腔注射重新麻醉鼠标。测量耳朵厚度之前。 </li><li>应用12.5微升1.25％OX的到右耳的两侧。申请12.5微升的丙酮/橄榄油载体溶液到左耳的每一侧。 </li><li>保持鼠标从笼子里的队友分开直到完全恢复通过其他老鼠，防止侮辱它的耳朵，然后在其原来的笼子代替鼠标，返回畜舍设施。 注:脱毛可能会导致轻微的皮肤炎症的耳朵厚度的变化之后。这轻微的炎症会3天后决心在正常耳厚度证实。 24小时局部应用后，OX-治疗耳显示显著（P &lt;0.01）相比消肿载体溶液单独处理的耳朵。 </li></ol>在pIL1-红色荧光蛋白的小鼠中性粒细胞3.标签注: 在低剂量˚F嗜中性粒细胞的体内标记luorescence，结合具体的中性粒细胞单克隆抗体遵循最近开发协议19。眼眶后注射根据具有一些修改24已建立的方案进行。 <ol><li>通过在90微升磷酸盐缓冲盐水（PBS）的稀释10微升的0.5毫克/毫升的Alexa Fluor 647缀合的抗Ly6G单抗（克隆1A8）制备抗Ly6G单抗工作溶液。 </li><li>转移100μl的抗Ly6G单抗工作液（50微克/毫升）的与28号针头的U-100胰岛素注射器。 </li><li>麻醉由先前描述的麻醉剂鸡尾酒（2.1）腹腔注射成年pIL1-红色荧光蛋白鼠标。放置鼠标麻醉腹部倒在一个干净的工作板。 </li><li>适用于温和的下行压力对皮肤的背部和腹部的一个鼠标眼睛凸出部分从插座眼球。 </li><li>小心地将针，斜面向下，以大约30°的角度在内侧眼角到眼窝窦。 注:操作员可以感到有压力，渗透和减轻阻力，一旦针头插入到窦。 </li><li>慢慢地，顺利地注入100微升抗Ly6G单抗工作液（5微克单抗/鼠标/注射）到鼠标。迅速取出针。出血有少量表明成功注入。 </li><li>立即申请1.25％OX和车辆解决方案到左，右鼠标的耳朵。 </li><li> 8小时后，管理100微升通过眶后注入新鲜烹制的抗Ly6G单抗工作液（5微克单抗/鼠标/注射）到同一个鼠标。 </li><li>为了形象化血管，立即成像前，辖100微升30毫克/毫升异硫氰酸荧光素（FITC）标记通过眼眶注射右旋糖酐（150 kDa的）。 </li></ol> 4.中性粒细胞引发的活体成像在弼1 - 红色荧光蛋白的小鼠<ol><li>麻醉鼠标麻醉鸡尾酒（2.1）的腹腔注射。0;适用兽医药膏鼠​​标的眼睛，以防止同时在麻醉状态下进行成像干燥。 <ol><li>通过稀释500μl的原始麻醉鸡尾酒与等体积的PBS制备1毫升半剂量麻醉剂鸡尾酒。 </li><li>半剂量的麻醉剂鸡尾酒转移至1毫升注射器与蝴蝶27号针头。 </li><li>将蝴蝶的针入小鼠腹腔腹部，并用胶带固定蝴蝶翅膀腹部。 </li></ol></li><li>放置一个玻璃盖玻片在成像阶段的​​中心。胶带将盖玻片的边缘以保持它在适当位置。 </li><li>放置的PBS一滴的小鼠耳的腹面以及上盖玻片。 </li><li>位置麻醉鼠标的耳朵在盖玻片。安装耳的末端，背侧向下，由夹在盖玻片与载玻片，通过胶带代替也保持之间的耳朵。 </li><li>打开多激光荧光共聚焦显微镜和所有相关的电子商务quipment。关掉房间的灯，以减少环境光曝光。 </li><li>打开图像采集软件。为检测选择了染料列表菜单荧光染料的激光通道。 </li><li>通过从血管和红色信号从通过目镜DsRed的+细胞绿色信号的观测调整焦距发炎耳朵皮肤上。 </li><li>与2微秒/像素的扫描速度和512×512像素的分辨率执行快速扫描。选择在实时视图菜单中每个通道伪。调整对焦旋钮设置结束，开始扫描的位置。 </li><li> 8微秒/像素和800×800或1024×1,024像素的分辨率 - 与4的扫描速度慢进行扫描。调整高电压，增益，并且在每个单独的信道偏移，最大化的信号的强度，同时避免饱和（红色像素）。应只在每个信道的几个红色像素。 </li><li>通过扫描次创建三维图像集Ë耳部皮肤角质层表面开始，以X，Y，317微米×317微米×30微米（40倍物镜）z的卷向下推进，635微米×635微米×30微米（20X物镜），或1270微米× 1270微米×50在2微米的Z-步骤微米（10X的目标）。 注:角质层是表皮的最外层，并且可以容易地本地化是根据它的强的自体荧光信号。 </li><li>在时间推移成像实验中，记录的三维图像，每2或4分钟长达8小时。 </li><li>通过蝴蝶针10微升半剂量麻醉鸡尾酒 - 间断，小鼠开始从麻醉中恢复，悉基于抽搐晶须观测，施用5。 注:请谨慎麻醉剂量 - 过量可能会导致鼠标死亡。可替代地，在一个罐子吸入麻醉可以应用于鼠标短期麻醉和鼻锥可用于长期成像。 </li><li>从舞台中删除麻醉鼠标时成像完成。取下胶带和鼠标的腹部取出蝴蝶针。返回鼠标在动物壳体设施单独笼子。 注: - 3小时对于短期成像（&lt;1小时），小鼠完全从麻醉迅速1中恢复。对于长期成像（〜8小时），将小鼠恢复缓慢，12的一个典型的恢复期间 - 16小时。因此，口服水行政至少几次复苏期间，以防止严重脱水建议。 </li><li>过程成像数据集，跟踪单个细胞，生成洄游路径，并使用图像分析软件25计算细胞迁移的方向性和速度。 </li></ol>

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Pharmacol. 127 (8), 1915-1921 (1999).</li><li>Levin, M., Leibrecht, H., Ryan, J., Van Dolah, F., De Guise, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+domoic+acid+differ+between+in+vivo+and+in+vitro+exposure+in+mice.">Immunomodulatory effects of domoic acid differ between in vivo and in vitro exposure in mice.</a> Mar. Drugs. 6 (4), 636-659 (2008).</li><li>Basu, S., Hodgson, G., Katz, M., Dunn, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+role+of+G-CSF+in+the+production,+survival,+and+release+of+neutrophils+from+bone+marrow+into+circulation.">Evaluation of role of G-CSF in the production, survival, and release of neutrophils from bone marrow into circulation.</a> Blood. 100 (3), 854-861 (2002).</li><li>Kreft, M., Stenovec, M., Zorec, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focus-drift+correction+in+time-lapse+confocal+imaging.">Focus-drift correction in time-lapse confocal imaging.</a> Ann. N. Y. Acad. Sci. 1048, 321-330 (2005).</li><li>Zucker, R. 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Dermatol. 134 (11), 2680-2684 (2014).</li><li>Egawa, G., Natsuaki, Y., Miyachi, Y., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+epidermal+keratinocytes+and+dermal+vasculatures+using+two-photon+microscopy.">Three-dimensional imaging of epidermal keratinocytes and dermal vasculatures using two-photon microscopy.</a> J. Dermatol. Sci. 70 (2), 143-145 (2013).</li><li>Kedrin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nat. Methods. 5 (12), 1019-1021 (2008).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+2-photon+imaging+of+blood+flow+in+the+neocortex+through+a+cranial+window.">A method for 2-photon imaging of blood flow in the neocortex through a cranial window.</a> J. Vis. Exp. (12), (2008).</li><li>Ritsma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+implantation+of+an+abdominal+imaging+window+for+intravital+microscopy.">Surgical implantation of an abdominal imaging window for intravital microscopy.</a> Nat. Protoc. 8 (3), 583-594 (2013).</li><li>Looney, M. 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The authors have no financial conflicts of interest.

本研究的目的是开发一种技术用于监测中性粒涂刷的过程在活的动物，这尚未得到满足由目前可用的技术。为了实现这一目标，三既定方法被执行:1）皮肤炎症的诱导IL-1β的启动子驱动的DsRed的报告小鼠作为涂刷的量度，用低剂量的荧光缀合的抗Ly6G的嗜中性粒细胞的2） 体内标记单克隆抗体，以及3）活体共聚焦显微镜成像。这三种方法的组合使炎性皮损致敏中性粒细胞（红色荧光蛋白+</...

中性粒细胞在流通中最丰富的，短命的白细胞。他们迅速招募到感染或损伤部位，在那里他们通过与含有抗菌肽和蛋白酶1粒沿着活性氧和氮中间体的发布作为专业的吞噬细胞。在他们的招聘，中性粒细胞"引"的各种试剂，包括微生物产物，趋化因子和炎症细胞因子，从而在抵达机场时明显增强吞噬细胞的功能在炎症2的网站。嗜中性粒细胞引发的机制已被广泛研究的体外 3,4;然而， 在体内的过程的动态监控一直不可能日期。 最近，活体成像已成为用于可视化和量化生物过程的细胞动力学在活生物体的重要技术。 IntraviTAL成像可通过常规的单光子激发显微术来进行（ 例如，共焦）或多光子显微镜接近5。随着时间的推移，大量的改进已在该技术实现更高的图像分辨率，改善成像深度达到，组织光损伤和增强防抖6,7下降。鉴于其独特的，以使随着时间的推移细胞迁移和互动的动态可视化能力，活体显微镜已广泛应用于免疫学8应用研究的不同领域。活体成像使免疫​​学家更好地理解和背景情况在活的动物模型在细胞和分子水平上既免疫应答。 在转基因的最新进展以及敲入报告小鼠已用于监测中性粒细胞的动力学行为活体动物提供了有用的工具。溶菌酶M助驱动的增强型绿色荧光蛋白敲入小鼠已被广泛使用在各种炎性过程，包括外渗，细菌感染，和无菌炎症9-15来表征嗜中性粒细胞，单核细胞和巨噬细胞的运动性。此外，表达细胞质荧光共振能量转移的生物传感器的转基因小鼠已在发炎肠16内研究嗜中性粒细胞细胞外调节的有丝分裂原激酶和蛋白激酶A的活动使用。以高特异性的鼠模型用于荧光表达在嗜中性粒细胞是追赶敲入小鼠，其产生Cre重组以及荧光蛋白质tdTomato，其本身被耦合到淋巴细胞抗原6G（Ly6G）17的表达。从这个模型Ly6G缺陷嗜中性粒细胞的可视化已经证明，这些细胞中的各种无菌或感染体内炎症上下文发挥正常功能。表达红色荧光蛋白荧光p的转基因小鼠认为包括嗜中性粒细胞，炎性单核细胞，和活化的巨噬细胞 - - interleuikin-1β（IL-1β）启动子（pIL1-DsRed的）已被用于可视化的IL-1β产生细胞的能动行为鼠标的控制下rotein基因新兴在发炎的皮肤18。 体内标记可作为跟踪的中性粒细胞的细胞和分子的行为在炎症组织的另一种方法。低剂量荧光的静脉注射标记的抗Gr-1单克隆抗体（mAb），GR-1 +嗜中性粒细胞的募集级联后已被显现在小鼠皮肤损伤感染金黄色葡萄球菌 19。 体内含有链霉抗偶联物的施用缀705纳米的量子点和生物素化抗Ly6G单抗特异性标记循环嗜中性粒细胞20。此外，这样的共轭物成neutroph的内吞IL囊泡允许在嗜中性粒细胞迁移进入间质高速囊泡运输的跟踪。 体内用抗P-选择荧光标记的抗体的糖蛋白配体-1（PSGL-1），L-选择素（CD62L），整联αM标记（CD11b的）和趋化因子（CXC母题）受体2（CXCR2）在TNFα诱发炎症模式在早期的炎症已经21阐明在发挥作用的调节机制。偏振中性粒凸出PSGL-1富含腹足上活化的血小板与CD62L本交互，导致CD11b和CXCR2，驱动中性粒细胞迁移和引发炎症受体的再分配。 IL-1β是在致敏嗜中性粒细胞22升高签名基因之一。在pIL1-DsRed的报告小鼠，红色荧光蛋白的荧光信号（ 即 ，IL-1β启动子活化）与IL-1β的mRNA表达和IL-1β蛋白质的生产正相关。&lt;SUP&gt; 18为了监测中性粒涂刷的过程中，一个活体显微镜方法的开发涉及在pIL1-DsRed的小鼠模型皮肤炎症的诱导恶唑酮（OX）以下的中性粒细胞的荧光缀合的抗Ly6G单抗体内标记。通过这种模式，能够研究在各种疾病和病症的动物模型中引发的中性粒细胞的行为和功能。

<table><tbody><tr><td>Heparin sodium</td><td>APP Pharmaceuticals</td><td>NDC 63323-540-31</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Lonza</td><td>10-548E</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich</td><td>F0926</td><td></td></tr><tr><td>Lipopolysaccharides</td><td>Sigma-Aldrich</td><td>L4391</td><td></td></tr><tr><td>Ketamine hydrochloride</td><td>Hospira</td><td>NDC 0409-2051-05</td><td></td></tr><tr><td>Xylazine</td><td>LLOYD Laboratory</td><td>NADA #139-236</td><td></td></tr><tr><td>Acepromazine</td><td>Boehringer Ingelheim</td><td>ANADA 200-361</td><td></td></tr><tr><td>Hair-removal cream</td><td>Church &amp;amp; Dwight</td><td></td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A16P4</td><td></td></tr><tr><td>Oxazolone</td><td>Sigma-Aldrich</td><td>E0753</td><td></td></tr><tr><td>Alexa Fluor 647 anti-mouse Ly6G antibody</td><td>BioLegend</td><td>127610</td><td></td></tr><tr><td>U-100 insulin syringe with 28 G needle</td><td>BD</td><td>329461</td><td></td></tr><tr><td>FITC-CM-Dextran, 150 kDa</td><td>Sigma-Aldrich</td><td>74817</td><td></td></tr><tr><td>Butterfly infusion set (27 G needle)</td><td>BD</td><td>387312</td><td></td></tr><tr><td>FACSCalibur cytometer</td><td>BD</td><td></td><td></td></tr><tr><td>CellQuest Pro software</td><td>BD</td><td></td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Metamorph Software</td><td>Universal Imaging</td><td></td><td></td></tr></tbody></table>

intravital imaging of neutrophil priming using il-1&#946; promoter-driven dsred reporter mice

中性粒细胞是人体血液循环中最丰富的白细胞，并迅速募集到炎症部位。底漆是增强中性粒细胞的吞噬功能的重要事件。虽然大量研究纷纷亮相感染和损伤过程中存在的和中性粒细胞引发的重要性，意味着可视化这一进程的体内已经不可用。所提供的协议使嗜中性粒细胞在活的动物引发的动态过程的监测通过组合三种方法:1）红色荧光蛋白报告信号-作为吸2） 在体内嗜中性粒细胞标记的量度-通过荧光标记的抗淋巴细胞抗原的注射来实现6G（Ly6G）单克隆抗体（mAb）和3）活体共焦成像。几个关键步骤都参与了这一协议:恶唑酮诱导小鼠耳部皮肤炎症，动物适当镇静，抗Ly6G单抗重复注射，和prev成像过程中的焦点漂移ention。虽然有一些限制已经观察到，如连续成像时间（〜8小时）在一只小鼠的限制和异硫氰酸荧光素 - 葡聚糖从血管中的炎症状态的泄漏，该协议提供用于活体成像的基本框架底漆嗜中性粒细胞的行为和功能，可以很容易地扩展到在小鼠炎症模型其他免疫细胞的检查。

pIL1-DsRed的小鼠的筛选是根据通过使用流式细胞仪的外周血白细胞产生的表型DsRed的荧光信号进行。 LPS刺激已知诱导IL-1β生产的骨髓细胞包括中性粒细胞，单核细胞和树突状细胞26-28。因此，分离的白细胞培养用LPS用于向流式细胞仪分析前4小时。接着，门被设置在这个门控群体测量循环基于小区大小（FSC）和内部的复杂性（SSC）（ 图1A）29，和...

Watch this Scientific Journal Video about Intravital Imaging of Neutrophil Priming Using IL-1Î² Promoter-driven DsRed Reporter Mice at JoVE.com

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

intravital-imaging-neutrophil-priming-using-il-1-promoter-driven

Research

JoVE Journal

Immunology and Infection

9.5K 次观看.  University of Toledo College of Medicine and Life Sciences. This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals. 

视频: Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

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

白细胞的归位和迁移的非侵入性的成像在体内</em

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

科研

免疫与感染

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

通过体内显微镜评估的小鼠中白细胞肌渗透

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Detecting Migration and Infiltration of Neutrophils in Mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

检测小鼠中性粒细胞的迁移和渗透

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

生物学

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

对小鼠皮肤中的中性粒细胞迁移进行成像，以研究生理条件下的亚细胞膜重塑

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by imaging.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

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白细胞的归位和迁移的非侵入性的成像在体内