这项研究得到了韩国政府资助的韩国国家研究基金会 (NRF) 的一笔赠款 (MSIP; 对 Y-m 的赠款2016R1A2B4008199H.) 和大韩民国卫生 & 福利部 (赠款号: HI14C1324)。

所有程序都是按照韩国世大学医学院机构动物保育和使用委员会的指导方针进行的。LysM-绿色荧光蛋白 (gfp; gfp/+) 老鼠8和 CX3Cr1-GFP (gfp/+)9小鼠在8-12 周龄时被用于活体成像。所有的手术工具都是用70% 酒精蒸压和消毒的。
1. 具有辅助工具的定制设计的小鼠肝脏成像室
<ol><li>小鼠肝脏成像室设计<ol>
<li>为小鼠肝脏成像 (图 1) 制作如下尺寸的腔室: 内腔 (120 mm 长度 x 160 mm 宽度 x 30 mm 高度), 外腔 (100 mm 长度 x 100 mm 宽度 x 24 mm 高度), 螺栓 (Ф 3 mm x 30 mm 高度), 螺母 (Ф 4 mm)和翅形螺母 (Ф 4 mm)。</li>
		<li>测量固化剂和弹性体基在1:9 的比例, 使共120克硅。</li>
		<li>将液体硅胶混合物放入250毫升锥形瓶中, 并将其混合均匀。然后把烧瓶放进干燥, 在真空泵的真空下, 将气泡取出约1小时。</li>
		<li>倒入液体硅胶混合成一个定制设计的老鼠室高达10毫米的高度, 让它治愈了几天 (图 1A)。</li>
	</ol>
</li>
	<li>覆盖片和肝固定床的制备<ol>
<li>在金属框架上, 用硅胶胶水把盖子玻璃贴上。让它干1天 (图 1B)。</li>
		<li>为了有效地控制水浸透镜的蒸馏水, 一个永久性的堵水结构是必不可少的。使 1 mm 四舍五入硅胶胶水和硬化过夜。</li>
		<li>混合固化剂和弹性体基础, 然后倒入一个6孔板在一个高度约8-10 毫米. 执行硬化过程, 如所述, 在 1.1. 2-1. 1.4。</li>
	</ol>
</li>
</ol>2. LPS 的制备
<ol><li>制备无菌1x 磷酸盐缓冲盐水 (PBS), 以稀释从沙门氏菌 enterica血清型肠炎的 LPS 粉末。(1x PBS: 137 毫米氯化钠, 2.7 毫米氯化钾, 10 毫米 Na2HPO4, 1.8 mm,2PO4)。</li>
	<li>预热 PBS 在37° c 水浴, 并仔细打开 LPS 粉末瓶 (25 毫克)。</li>
	<li>使用吸管的援助, 倒入无菌 PBS (5 毫升) 到 LPS 粉瓶, 并轻轻搅拌或暂停解决方案, 准备库存解决方案。 注: 在 LPS 粉末液化后, 苛刻的涡流会引起气泡。在1.5 毫升的管子中制造等分时, 避免气泡使损失降到最低。</li>
	<li>储存在20° c 的股票 lps 溶液, 并将其稀释为1毫克/毫升 lps 溶液注射。</li>
	<li>使用1毫升注射器将1毫克/毫升 LPS 溶液 (20 毫克/千克) 注入非麻醉小鼠的腹膜中。对于控制动物, 注入相同体积的 PBS。</li>
	<li>等待2小时足够的炎症反应。</li>
</ol>3. 得克萨斯红葡聚糖用于染色血管的制备
<ol><li>准备两个15毫升锥形管和10毫升无菌 PBS。</li>
	<li>在3毫升的 PBS 中稀释得克萨斯红葡聚糖粉 (25 毫克), 并将其混合均匀。将混合物转移到一个空的15毫升锥形管。</li>
	<li>将剩余的 PBS (7 毫升) 转移到含有葡聚糖混合物 (3 毫升) 的15毫升锥形管中。</li>
	<li>将0.45 µm 注射器过滤器连接到10毫升注射器的尖端。注射器过滤器应该替换针。</li>
	<li>过滤葡聚糖混合物 (10 毫升)。均匀按压注射器以避免溢出。</li>
	<li>500µL 2.5 毫克/毫升右旋糖酐溶液在轻堵塞的1.5 毫升管和存储在20° c。解冻后, 将其储存在4° c。</li>
</ol>4. 手术过程
<ol><li>在手术前用70% 酒精消毒手术台和所有器械。然后, 设置用于肝脏手术的工具, 并将加热板调整到37° c (图 2A, B)。</li>
	<li>对于麻醉, 腹腔注射30毫克/公斤的 tiletamine/唑和10毫克/公斤的嗪。这些解决方案可以混合和稀释在 PBS 的同时注射。通过轻轻按压鼠标的脚趾来确认麻醉。 当没有反射时继续下一步。</li>
	<li>用少量的眼膏来预防小鼠视网膜的干燥。</li>
	<li>使用脱毛膏 (图 2C) 在腹部周围移除体毛。</li>
	<li>注入200µL 的得克萨斯红葡聚糖溶液 (浓度2.5 毫克/毫升) 通过尾静脉或复古轨道注射使用胰岛素注射器 (图 2D)。</li>
	<li>用标记标记右下区域。这条线应该从剑的过程到肋骨的末端。用钳子和剪刀切开表皮 (图 2E, F)。</li>
	<li>用微剪刀和镊子打开腹腔, 仔细地暴露肝脏。使用棉签 (图 2G, H) 将左外侧叶滚出。</li>
	<li>将500µL 的无菌 PBS 放在腹腔内, 防止肝脏干燥。</li>
	<li>将鼠标放在一个定制设计的房间的右卧位位置, 并在硅床上涂抹少量的组织胶。然后, 用棉签仔细地将肝脏附着 (图 2I)。 注: 由于肝组织极其易碎, 不机械拔肝。用棉签轻轻地滚出来。</li>
	<li>几秒钟后, 再用500µL 的无菌 PBS 湿肝, 避免肝脏干燥。将金属框架放置在肝脏上, 并将其固定在螺母上 (图 2J)。</li>
</ol>5. 双光子显微镜对肝脏的活体成像
<ol><li>运行禅宗软件 (卡尔蔡司) 打开激光, 设置波长为 880 nm, 并设置绿色, 和红色通道。根据荧光强度调整激光功率 (图 3A)。发射过滤器信息如下: Green/Alexa488, 500-550 毫微米和红色或 Alexa 555, 575-610 毫微米。</li>
	<li>将腔室置于成像阶段, 并将20X 水浸泡物镜靠近盖板玻璃。</li>
	<li>将硅胶加热器固定在鼠标体内以保持其体温 (图 3B)。</li>
	<li>把500µL 的蒸馏水放在金属架上, 然后将物镜靠近肝脏 (图 3C)。</li>
	<li>调整焦点并确定成像区域的深度和时间。</li>
	<li>进行成像。</li>
	<li>对于长期成像, 在成像过程中每小时注入半剂量的 tiletamine/唑和嗪合剂。在这一阶段, 保持37° c 小鼠的体温是必不可少的。始终保持对鼠标的眼睛, 因为麻醉时间不同于每一个鼠标。 注: 此方法不适用于术后恢复及影像学检查。我们在手术后立即牺牲鼠标。</li>
	<li>检查踏板反射每30分钟的稳定麻醉。</li>
</ol>6. 数据分析
<ol><li>打开数据分析软件 Volocity, 然后将库中的原始数据导入到软件中。</li>
	<li>从图像中删除噪音以适应情况并拍摄快照。 注意: 要获得平滑图像, 请使用 "精细" 或 "中位" 滤镜。此外, 使用 "对比度增强" 功能使深色或模糊图像更清晰。</li>
	<li>将编辑的项目调整为 100%, 并以 JPEG 或 TIFF 文件的格式导出为电影的快照图像和 AVI 文件。</li>
</ol>

<ol>
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作者声明没有竞争的金融利益。

由于其功能的重要性, 许多组3,10都积极研究了肝脏。例如, Heymann et al. 提出了一种微妙的方法使用琼脂糖。虽然这种方法被认为是高度精确的, 琼脂糖设置需要时间。然而, 用我们的方法, 所有的程序可以在30分钟内进行, 尽量减少其他混杂因素。第二, 稳定肝脏是非常重要的, 因为心跳会扰乱视频。为了消除这种现象, 我们将小鼠重新定位到合适的卧位位?...

肝脏是哺乳动物的主要代谢器官;它充当能量、荷尔蒙和解毒的控制塔1。由于其重要性, 研究人员已经进行了许多体外和体内实验来阐明炎症过程中肝脏的变化。活体显微镜已被用来捕捉活体图像从肝脏2 。的确, 各种肝脏成像方法已经被引入2,3,4,5,6。然而, 为了发展一个更简化的手术方法, 并实现目标器官和免疫细胞的稳定, 我们设计了一个简单的协议, 可以指导研究人员减少他们的活体成像的努力。
在本研究中, 我们介绍了一个稳定和新的成像室和外科技术, 可用于减少出血和可能感染。我们证实肝脏保持完好超过2小时。利用这种方法, 我们成功地确定了 LCMs7的形态和脓毒性条件下的中性粒细胞。由于这种方法最大限度地减少物理和生物混杂因素, 如颤抖和意外炎症在外科手术过程中, 我们预计, 这种方法可以用来观察急性反应的免疫细胞在肝脏响应像 LPS 的试剂

<table border="0" cellpadding="0" cellspacing="0" width="1004">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Custom designed chamber</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Custom-designed size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Metal cover slide</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Custom-designed size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cover glass</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td style="width:272px;">#72204-03</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sylgard 184 Silicone elastomer kit</td>
			<td style="width:239px;">Dow Corning</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Erlenmeyer flask</td>
			<td style="width:239px;">DURAN</td>
			<td style="width:272px;">21 216 36</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cell culture plate (Flat type)</td>
			<td style="width:239px;">HYUNDAI Micro Co.</td>
			<td style="width:272px;">H31006</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Desiccator</td>
			<td style="width:239px;">ADARSH</td>
			<td style="width:272px;">55204</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">High Q BOND SE 5 mL</td>
			<td style="width:239px;">BJM LAB</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">To make semi-permanent dam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Flexible Silicone Rubber Heaters</td>
			<td style="width:239px;">Omega</td>
			<td style="width:272px;">SRFR/SRFG SERIES, M1250/0800</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">DC power supply</td>
			<td style="width:239px;">Toyotech</td>
			<td style="width:272px;">DP30-03A</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Phosphate-buffered saline</td>
			<td style="width:239px;"></td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Home-made</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:260px;">Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:272px;">L6011</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Texas Red Dextran</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:272px;">D1830</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:260px;">15 mL High-clarity polypropylene 
			conical tube</td>
			<td style="width:239px;">FALCON</td>
			<td style="width:272px;">14-959-49B</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:260px;">0.45 &#956;m filter (Minisart Syringe Filter)</td>
			<td style="width:239px;">Sartorius</td>
			<td style="width:272px;">16555k</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1.5ml Micro Tube, Amber</td>
			<td style="width:239px;">Axygen</td>
			<td style="width:272px;">MCT-150-X</td>
			<td style="width:233px;">For light-blocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1 mL syringe</td>
			<td style="width:239px;">Korea Vaccine</td>
			<td style="width:272px;">KV-S01</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">3 mL syringe</td>
			<td style="width:239px;">Korea Vaccine</td>
			<td style="width:272px;">KV-S03</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">10 mL syringe</td>
			<td style="width:239px;">Korea Vaccine</td>
			<td style="width:272px;">KV-S10</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">0.3 mL insulin syringe</td>
			<td style="width:239px;">Becton Dickinson</td>
			<td style="width:272px;">324900</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Hair removal cream 100 mL</td>
			<td style="width:239px;">Body natur</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cotton swab</td>
			<td style="width:239px;">ABDI</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Zoletil 50 5 mL</td>
			<td style="width:239px;">Virbac</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rompun 10 mL</td>
			<td style="width:239px;">Bayer</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Heating plate</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;">PH-S-10</td>
			<td style="width:233px;">Custom-designed size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">DC controller</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;">CU-301</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Microdessection Scissors</td>
			<td style="width:239px;">Harvard Apparatus</td>
			<td style="width:272px;">72-5418</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Scissors</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:272px;">RS-5882</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Forceps</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:272px;">RS-5137</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Vet bond</td>
			<td style="width:239px;">3M</td>
			<td style="width:272px;">1469SB</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">ZEN software (Black Edition)</td>
			<td style="width:239px;">Carl Zeiss</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Program for LSM 7 MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">LSM 7 MP</td>
			<td style="width:239px;">Carl Zeiss</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Volocity</td>
			<td style="width:239px;">PerkinElmer</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Analysis program</td>
		</tr>
	</tbody>
</table>

two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver

脓毒症是一种严重的感染, 可能导致器官衰竭和组织损伤。虽然与脓毒症有关的死亡率和发病率极高, 但没有对直接治疗或与器官有关的机制进行详细的实时检查。肝脏是管理人体内毒素和感染的关键器官。本文旨在对内毒素血症诱导后的小鼠肝脏进行活体成像, 以跟踪免疫细胞的运动, 如中性粒细胞和肝囊巨噬细胞 (LCMs)。因此, 我们设计了一种新的手术方法, 以接触肝脏微创手术。小鼠腹腔注射脂多糖 (LPS), 一种常见的内毒素。利用我们的新的手术方法, 接触和活体成像的小鼠肝脏, 我们发现, 中性粒细胞招募在 LPS 治疗 LysM 绿色荧光蛋白 (GFP) 小鼠肝脏增加与磷酸盐缓冲生理盐水治疗的肝脏。LPS 治疗后, 中性粒细胞数量随着时间的推移显著增加。此外, 使用 CX3Cr1-GFP 小鼠, 我们成功地可视化肝居民巨噬细胞称为 LCMs。因此, 为了研究新的试剂对控制免疫移动性的有效性在体内, 确定中性粒细胞和 LCMs 在肝脏中的运动和形态学, 可以让我们发现在器官衰竭和组织损伤中所造成的治疗作用白细胞在脓毒症中活化。

一个简单和非常稳定的成像室设计。室的底部布满了硅胶, 防止滑鼠的身体和器官。此外, 由于硅具有适当的弹性, 它可以防止因盖板滑动 (图 1A、 C) 的压力而引起的预期损坏。其次, 采用组织安全胶 (兽医键) 固定在环形硅胶床上提取肝脏。这种方法使我们能够避免因心跳或呼吸运动而引起的颤抖。最后, 根据小鼠的身体尺寸和突起肝脏的...

Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

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

我们建立了一种新的小鼠肝脏双光子成像的手术方案。利用这一技术, 我们确定了肝内毒素诱导中的内毒素血症的详细结构。我们预期这种方法可以用来确定各种试剂治疗肝白细胞迁移的有效性。

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

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

two-photon-intravital-imaging-leukocytes-during-immune-response

Research

JoVE Journal

Immunology and Infection

10.1K 次观看.  Yonsei University College of Medicine. 我们建立了一种新的小鼠肝脏双光子成像的手术方案。利用这一技术, 我们确定了肝内毒素诱导中的内毒素血症的详细结构。我们预期这种方法可以用来确定各种试剂治疗肝白细胞迁移的有效性。

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

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

科研

免疫与感染

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

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

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

活体成像的鼠标使用2 - 光子显微镜的胸腺

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

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform 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 ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染:肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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

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

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

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

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

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

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

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

双光子激光显微镜在细胞膜修复中的应用

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

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

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寄生蜂简介

活体成像的鼠标使用2 - 光子显微镜的胸腺

小鼠腘窝淋巴结的活体成像

定量测定的免疫反应和睡眠模式

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

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