Name: intravital imaging of neutrophil priming using il-1&#946; promoter-driven dsred reporter mice
Uploaded: 2016-06-22T13:00:00
Description: Watch this Scientific Journal Video about Intravital Imaging of Neutrophil Priming Using IL-1Î² Promoter-driven DsRed Reporter Mice at JoVE.com

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?...

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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。

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		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Heparin sodium</td>
			<td style="width:165px;">APP Pharmaceuticals</td>
			<td style="width:139px;">NDC 63323-540-31</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">ACK lysing buffer</td>
			<td style="width:165px;">Lonza</td>
			<td style="width:139px;">10-548E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Fetal bovine serum</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">F0926</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Lipopolysaccharides</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">L4391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Ketamine hydrochloride</td>
			<td style="width:165px;">Hospira</td>
			<td style="width:139px;">NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td style="width:165px;">LLOYD Laboratory</td>
			<td style="width:139px;">NADA #139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acepromazine</td>
			<td style="width:165px;">Boehringer Ingelheim</td>
			<td style="width:139px;">ANADA 200-361</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair-removal cream</td>
			<td style="width:165px;">Church &#38; Dwight</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:139px;">A16P4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxazolone</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">E0753</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:196px;">Alexa Fluor 647 anti-mouse Ly6G antibody</td>
			<td>BioLegend</td>
			<td style="width:139px;">127610</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:196px;">U-100 insulin syringe with 28 G needle</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">FITC-CM-Dextran, 150 Kda</td>
			<td>Sigma-Aldrich</td>
			<td style="width:139px;">74817</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:196px;">Butterfly infusion set (27 G needle)</td>
			<td>BD</td>
			<td style="width:139px;">387312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCalibur cytometer</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellQuest Pro software</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

The overall goal of this procedure is to present a technology for monitoring the process of neutrophil priming in living animals. This method can help answer the key questions in the immunology field, such as visualization of the behavior and the function of priming the neutrophils in animal models of rarest diseases and disorders. The main advantage of this technique is that it combines three established methodologies, including fluorescent reporters in viral labeling and intravital imaging.Breed heterozygous pIL1-DsRed mice with wild-type C57Black six mice. At three to four weeks, phenotype their pups. For each pup, prepare a 1.5 milliliter tube with 20 microliters of heparin.To collect blood, hold a pup by its neck scruff and pierce the submandibular vein in the cheek pouch using a 5 millimeter lancet. Only a small puncture is needed. Collect three to five drops of blood per tube.After making the collection, be sure to stop the bleeding and observe the animals for 30 minutes before returning them to the animal facility. To each sample tube, add 500 microliters of red blood cell lysing buffer. Then, vortex the tubes, and incubate them for five to 10 minutes on ice.After the incubation, slowly add 400 to 500 microliters of fetal bovine serum under the cell suspensions in each tube. A clear interface should form between the layers. Next, centrifuge the samples at 1, 500 Gs for five minutes to collect cell pellets.Discard the supernatent and resuspend the cells in 200 microliters of complete RPMI-1640 medium. Then, transfer each suspension to a flow cytometry tube and add 20 microliters of freshly made LPS Working Solution to each tube. Incubate the samples for four hours before performing flow cytometry.To begin, anesthetize the mouse with an intraparetoneal injection of an anesthetic cocktail and confirm adequate sedation with a toe pinch test. Next, apply hair removal cream to the dorsal surface of both ears, wipe it off within a minute, and wash the skin with water. Measure the thickness of the ear where the applications were made using a micrometer.Later, return the mouse in its own cage after it has recovered from the anesthesia, and allow the mouse to rest for at least three days before further testing. After three days, reanesthetize the mouse and measure the ear thickness again to make sure it is not irritated. Then, apply 12.5 microliters of 1.25%oxazolone onto each side of the right ear, and apply 12.5 microliters of the acetone olive oil vehicle solution onto each side of the left ear.As always, observe the mouse recover from the anesthesia. 24 hours later, measure the thickness of the ear again under anesthesia to ensure the oxazolone induced skin inflammation has occurred. To begin, transfer 100 microliters of anti-lyse XG monocolonal antibody into a U100 insulin syringe with a 28 gauge needle.Next, anesthetize a treated mouse as previously described. With the mouse abdomen down, apply gentle downward pressure to the skin dorsal and ventral to an eye to partially protrude the eyeball. Then, carefully place the needle, bevel down, approximately 30 degrees to the medial canthus and carefully insert the needle into the retro orbital sinus.The penetration pressure will be relieved when the needle enters the sinus. Then, eject the content and remove the needle quickly. A small amount of bleeding suggests successful injection.Now, immediately apply 1.25%oxazolone and vehicle solution onto the right and left mouse ears as before. Eight hours later, administer a second dose of the same antibody into each mouse. Also, to visualize the blood vessels, retro orbitally administer 100 microliters of fitc dextran.For this procedure, first anesthetize the mouse as before. Then, apply veterinary ointment to the eyes to prevent dryness during imaging. Next, place a glass coverslip at the center of an imaging stage, and secure the edges of the coverslip with tape.Also, add a drop of PVS to the coverslip. Now, position the mouse's ear over the coverslip, dorsal side down. Next, moisten the ventral surface of the mouse ear with a drop of PVS.Then, mount the tip of the ear with a glass slide on top and secure the slide with tape. Now, turn on a multi laser fluorescence confocal microscope and the related equipment. Turn off the room lights to minimize ambient light exposure.Then, record three dimensional images using the confocal microscope. For time lapse imaging experiments, record three dimensional images every two to four minutes for up to eight hours. All the while, watch for twitching whiskers, which indicates that the mouse is beginning to recover from the anesthesia.In such situations, administer five to 10 microliters of a half dose of anesthetic cocktail via a butterfly needle. pIL-1-DsRed mice were screened for phenotypic DsRed fluorescence produced in peripheral blood leukocytes via flow cytometry. Circulating myeloid cells from wild-type mice minimally express DsRed, while the same cells from pIL1-DsRed mice express distinguishable DsRed signals.IL-1 beta promoter activation is a marker of neutrophil priming, and can be best visualized in living pIL-1DsRed mice. Next, a skin sensitizer, oxazolone, was topically applied to the right ear. Later, the neutrophils were labeled before and after a second application of the oxazolone sensitizer via retro orbital injection.Likewise, fitc dextran was injected to locate blood vessels. Many double labeled cells were found in the extravascular space, which represent primed neutrophils. A small number of cells were only labeled by the lyse XG antibody, which were probably resting neutrophils.Moreover, a small population of DsRed positive cells were observed, which may comprise inflammatory monocytes and activated macrophage. Cell migratory paths were tracked to compare the two neutrophil populations. Both neutrophil populations showed a random migration once the extracellular space had been entered.Interestingly, the putatively primed neutrophils exhibited a significantly higher velocity compared with their putatively resting counterparts. After its development, this technique paved the way for researchers in the field of immunology. Two explore the behavior and the function of many immune cells in various inflammatory states involving different t-cells.After watching this video, you should have a good understanding of how to monitor the dynamic process of neutrophil priming in living animals.

Research

JoVE Journal

Immunology and Infection

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

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 ...

科研

免疫与感染

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

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

Intravital Imaging of the Mouse Popliteal Lymph Node

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

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

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 ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

生发中心的高度已解决活体条纹照明显微镜

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

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

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, ...

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

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 ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

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 ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...