这项工作得到了马萨诸塞州总医院医学内部资金部（JMT，MKM的MLC，JMV），国家生物医学成像和生物工程授予T32EB006348（CEC），美国马萨诸塞州总医院的中心计算和综合生物学的发展基金和AI062773（ RJH），补助AI062773，DK83756，和DK 043351（RJX），0643745（MJL）美国国家科学基金会，美国国立卫生研究院R21CA133576（MJL），以及国家过敏和传染病研究所（NIAID的）美国国立卫生研究院（NIH）的AI057999（JMV ）。我们感谢尼古拉三尤德，有益的讨论和查尔斯毛毡（RPI的公司）提供技术援助。

1。光学诱捕病原体的文化条件<ol><li>生长在一个半固体琼脂的SBD（沙氏葡萄糖）媒体30 ° C 曲霉 （B-5233/RGD12-8）3天。 </li><li>成长C。白色念珠菌 （SC5314）在YPD（酵母蛋白胨葡萄糖）液体培养基在摇床培养箱含有100μg/ mL氨苄青霉素一夜之间30 ° C。 </li></ol> 2。病原体为荧光标记的制备<ol><li>收获所需量的病原体，并转移到1.5 ml反应管。 </li><li>反应管中加入300μL磷酸盐缓冲液（PBS）。 </li><li>超声为30秒的混合物。 </li><li>离心1分钟4000转。 </li><li>吸上清液，留下沉淀不受干扰。 </li><li>重复（2.4）和（2.5）两次以上。 </li><li>悬浮在500μL的PBS。 </li></ol> 3。用荧光染料标记的病原体<ol><li>溶解1毫克染料（如的Alexa Fluor 488的Alexa Fluor 647）100μL，二甲基甲酰胺（DMF）（浓度为10毫克/毫升）的兴趣。 </li><li>含洗病原体的反应管加入3μL染料混合物。 </li><li>旋转或摇晃样品在37 ° C为1小时。 </li><li>用PBS 3X洗1分钟4000转离心样品。 </li><li>悬浮于300μL的PBS。 </li></ol> 4。收获从全血T细胞<ol><li>获得全血（新鲜）。 </li><li>温血，PBS + 2％胎牛血清（FBS）和histopaque室温。 </li><li>在50μL/ ml全血加入RosetteSep人体的CD4 + T细胞富集酒会。 </li><li>旋转样品和20分钟在室温下孵育。 </li><li>稀释样品与等体积PBS + 2％FBS，轻轻混匀。 </li><li>层histopaque顶部稀释样品，最大限度地减少混合</li><li>离心20分钟，在室温下在1200 XG与制动关闭。 </li><li>删除丰富的细胞。 </li><li>丰富的细胞用PBS + 2％FBS的解决方案2X洗净。 </li><li>红细胞裂解红细胞裂解缓冲2分钟</li><li> 5分钟，加入10 mL PBS + 2％FBS和在1500转离心裂解红细胞中</li><li>吸上清，注意不要扰乱沉淀</li><li>悬浮在含10％胎牛血清的IMDM媒体</li></ol> 5。到玻片RAW 264.7巨噬细胞的制备<ol><li>准备贝科改良Eagle培养基）DMEM（含有10％FBS，1％青霉素/链霉素，L -谷氨酰胺和1％。 </li><li>暖媒体，胰蛋白酶，PBS在温水浴至37 ° C。 </li><li>用PBS清洗2倍板</li><li>吸之间每次洗的PBS。 </li><li>加入5 mL胰酶板盖面（10厘米组织培养板）。 </li><li> 5分钟在37 ° C。 </li><li>轻轻敲板块方面，从板面分离的细胞。要小心，不要溅板之外的胰蛋白酶。 </li><li>加入5 mL或同等数额的胰蛋白酶媒体。 </li><li>吸成一个反应管的混合物。 </li><li>在1000 XG离心3分钟。 </li><li>吸媒体，小心不要去打扰颗粒。 </li><li>悬浮在10毫升的媒体。 </li><li>加入400μL的媒体，每个腔室幻灯片。 </li><li>添加5μL的细胞悬液，每个分庭。 </li><li>培养箱中培养过夜增长37 ° C，5％的CO 2 。 </li></ol> 6。除了以样本的病原体<ol><li>吸取5-10μL利益标记的病原体（步骤1-3）进入会议厅。 </li><li>调匀pipeting向上和向下，小心不要触摸室底部扰乱坚持的巨噬细胞。 </li></ol> 7。样品装载到旋转盘共聚焦显微镜（视频，通过扫描组件） <ol><li>打开所有部件旋转盘共聚焦显微镜。 </li><li> DIC的成像显微镜对齐。 </li><li>从孵化器中删除室幻灯片。 </li><li>室幻灯片插入到专业舞台。 </li><li>卸下室幻灯片的顶部（DIC的成像必要）。 </li></ol> 8。光学诱捕的准备工作（视频，通过扫描组件和它是如何集成到显微镜） <ol><li>快门打开光陷阱。 </li><li>打开红外激光。 </li><li>打开快门（激光）光学陷阱。 </li><li>确认在前面的红外激光的快门是关闭的检查与IR卡。 </li></ol> 9。磁光阱的选择和操纵的病原体<ol><li>注重坚持幻灯片上的巨噬细胞。 </li><li>毗邻巨噬细胞在溶液中的自由波动的病原体。 </li><li>移动阶段，这样的病原体在陷阱附近。 </li><li>打开快门和从事陷阱。 </li><li>移动样品接触到的巨噬细胞与静止陷阱PED的病原体。 </li><li>图像与旋转盘共聚焦显微镜，DIC，荧光，或两者兼而有之。通常情况下，诱捕是在DIC，荧光实时成像。 </li></ol> 10。代表性的成果: 为了发挥的空间和时间的控制病原体和珠在体内和体外环境，我们设计一个定制的套色设备集成到一个旋转盘共聚焦显微镜（原理如图1） 。在充分的力量，激光提供350兆瓦电力，〜80兆瓦的电力在TIRF目标，用功率计测量，光耦合与各种光学元件在光学陷阱仪器后，形成的陷阱在会议厅内。 为了室相对被困对象的对象的位置，舞台很感动，而被困的对象与激光捕获静止。舞台是够慢的速度移动，使被困粒子的拖曳力不超过最大阱力。 白色念珠菌单独种群（典型的大小- 〜5微米），标有三种颜色（AF488，AF568，AF647，相应的绿色，蓝色和红色的身影，分别），说明多个荧光通道成像同时光学捕获的病原体。一个单一的C。白色念珠菌是被困在一个正方形图案通过其他酵母集群移动，灰色箭头表示，这表明能够捕获和操纵由运营商即使在拥挤的环境中选择一个单一的病原体的具体位置（图2A） 。 为了进一步说明这一制度陷阱病原微生物表现形态的不同形状的灵活性，光学镊子也能够保持和位于 A C. 白色念珠菌的粒子与一个假菌丝。 c. 在 AF647（红色）标记的白色念珠菌是沿着一个轨迹，所介绍的白色箭头，放在旁边的荧光GFP - LC3 - RAW细胞（图2B ）。酵母部分的C.白色念珠菌是被困的假菌丝沿着落后。 我们也定位曲霉下到原始的小鼠巨噬细胞，以分析与这一特定的细胞株和病原体（图3A）吞噬的绝对时限。一旦病原体与细胞接触，陷阱关闭，时间推移成像观察随后的细胞事件（图3B）。光陷阱也被用来捕捉主要的T细胞，从血液中分离出，并指示毗邻珠涂在为T细胞与抗CD3抗体，形成免疫突触与珠（图4），显示其他通用性，以直接捕获和操纵的免疫细胞。 <img src="/files/ftp_upload/3123/3123fig1.jpg" alt="图1"/> 图1概述和原理图相结合的光陷阱和旋转盘共聚焦显微镜设置。仪器布局诱捕束（紫色），明场成像（橙色），荧光激发光（红色），荧光（绿色），电荷耦合器件（CCD）照相机，道路照明电子倍增电荷耦合器件（EM CCD）相机，（D1和D2）分色镜，反射镜（M1和M2），透镜（L1，L2，L3，L4的）。图中的陷阱共聚焦显微镜系统的所有其他组件都标记。 <img src="/files/ftp_upload/3123/3123fig2.jpg" alt="图2"/> （一）荧光图像捕获和操纵C. 图2。 白色念珠菌 。被困荧光标记（的Alexa Fluor 488（AF488），蓝）生物体在其他C.荧光标记的领域白色念珠菌 （AF568，红，绿色和AF647）。舞台左右移动被困灰色箭头表示的，蓝色的CA粒子。 （二）受困伪菌丝形成的C. GFP - LC3的RAW细胞旁边白色念珠菌 。荧光标记的psuedohyphal形式的CA（红，AF647）光学捕获和移动相邻的GFP - LC3的表达RAW巨噬细胞。 CA的有机体是沿轨迹移动，白色箭头表示。 <img src="/files/ftp_upload/3123/3123fig3.jpg" alt="图3"/> 图3 A 的捕获和定位。 霉菌旁边的一个吞噬的RAW细胞。 （一）明场图像被困答霉菌 ，白色箭头指示，移动和沿红色箭头指示的路径的位置。被困的病原体是稍微重点推动略高于焦平面有机体的陷阱。 霉菌是移动，直到它被放在相邻的RAW细胞所需。 （二）流感A 的吞噬功能成像orescence 霉菌由RAW细胞。后被困的病原体是旁边放置到原始的细胞，吞噬过程中被激活。 30秒，原始细胞的细胞膜开始变化和粒子周围形成一个杯子。在60秒，一杯是完全形成。从90年代到150S，在A.霉菌是吞噬，180秒，粒子是完全内在<img src="/files/ftp_upload/3123/3123fig4.jpg" alt="图4"/> 图4。捕获的主要T细胞突触形成与抗CD3涂珠。一个主要的T细胞移动到一个位置，旁边与抗CD3抗体的磁珠涂光陷阱DIC图像。细胞然后形成免疫突触，不能在图像很容易看出端倪。

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在这项工作中，我们使用了一个光学陷阱捕捉到3微米之间的尺寸的病原体 - 5微米。虽然我们的实验室感兴趣的病原体通常有这些方面，这里所描述的光镊系统是灵活的陷阱大范围的大小。事实上，光学陷阱被用来捕捉范围从单个原子，细胞直径约10微米的颗粒。此外，这种光学诱捕系统能够捕捉到各种形状:球形的颗粒，椭圆形，而且非常细长的颗粒，这是有用的生物病原体时。 <p class="jove_con...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 曲霉 </td><td></td><td></td><td>白化菌株，B-5233/RGD12-8，KJ权涌，美国国立卫生研究院的礼物</td></tr><tr><td>白色念珠菌</td><td></td><td></td><td> SSY50 - B埃莱夫塞里奥Mylonakis，麻省总医院的礼物突变体; SC5314应变，从杰拉尔德芬克的礼物，怀特黑德研究所</td></tr><tr><td>的Alexa Fluor 488 </td><td> Invitrogen公司</td><td> A20000 </td><td></td></tr><tr><td>的Alexa Fluor 647 </td><td> Invitrogen公司</td><td> A20006 </td><td></td></tr><tr><td>二甲基甲酰胺</td><td>西格玛</td><td> D4551 </td><td></td></tr><tr><td>新鲜血液</td><td></td><td></td><td> RJW荒地，麻省总医院，HMS的礼物</td></tr><tr><td>尼康倒置显微镜</td><td>尼康</td><td></td><td>钛 - E型</td></tr><tr><td>补漏激光，ChromaLase </td><td>蓝天研究</td><td> CLAS - 106 - STF02 - 02 </td><td></td></tr><tr><td>荧光激发激光</td><td>连贯</td><td></td><td>模型伊诺70C </td></tr><tr><td>为诱捕组件的面包板</td><td> Thorlabs </td><td> MB1224，MB1218 </td><td></td></tr><tr><td>光学航空表</td><td>技术制造公司</td><td></td><td></td></tr><tr><td>踏板控制的电子快门</td><td> Uniblitz </td><td></td><td>购买文森特协会，纽约州罗彻斯特</td></tr><tr><td>单模光纤</td><td> OZ光学</td><td> PMJ - 3S3S - 1064 - 6 </td><td></td></tr><tr><td>光纤定位</td><td> Thorlabs </td><td> PAF - X - 5 - C </td><td></td></tr><tr><td>光纤准直器</td><td> OZ光学</td><td> HPUCO - 23 - 1064 - P - 25AC </td><td></td></tr><tr><td>为望远镜镜头</td><td> Thorlabs </td><td> AC254 - 150 - B </td><td>焦距为150毫米</td></tr><tr><td>翻译阶段（X，Y，Z） </td><td>纽波特</td><td> M - 461 - XYZ </td><td></td></tr><tr><td>红外分色镜</td><td>浓度</td><td> ET750 - SP - 2p8 </td><td></td></tr><tr><td>物镜（100X） </td><td>尼康</td><td></td><td> NA = 1.49，浸油，TIRF目标</td></tr><tr><td>共焦头</td><td>横河电机</td><td> CSU - 11 </td><td></td></tr><tr><td>偏光镜</td><td>尼康</td><td> MEN51941 </td><td></td></tr><tr><td> Wollaston棱镜</td><td>尼康</td><td> MBH76190 </td><td></td></tr><tr><td> EM - CCD摄像头</td><td>滨松</td><td> C9100 - 13 </td><td></td></tr><tr><td> CCD摄像头（ORCA的急诊室） </td><td>滨松</td><td> C4742 - 80 - 12AG </td><td></td></tr><tr><td>滤光轮</td><td> Ludl </td><td> 99A353 </td><td></td></tr><tr><td>滤光轮</td><td>萨特</td><td> LB10 -西北欧</td><td></td></tr><tr><td>墓室玻璃罩</td><td> Lab-Tek/Nunc </td><td> 155409 </td><td></td></tr><tr><td>磁珠</td><td> Invitrogen公司</td><td> 111 - 51D </td><td>涂有抗CD3 </td></tr><tr><td>贝科改良Eagle培养基（DMEM） </td><td> Invitrogen公司/ Gibco公司</td><td> 10313 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司/ Gibco公司</td><td> 15140-122 </td><td></td></tr><tr><td> L -谷氨酰胺</td><td> Invitrogen公司/ Gibco公司</td><td> 25030-081 </td><td></td></tr><tr><td>胎牛血清（Hyclone公司） </td><td> ThermoScientific </td><td> SH30071.03 </td><td></td></tr></tbody></table>

use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging

动态活细胞成像允许直接的免疫系统的1，2细胞之间的实时交互的可视化;然而，在空间和时间之间的吞噬细胞和微生物控制的缺乏已呈现集中观察到病原体的宿主反应的初步相互作用困难。从历史上看，如细 ​​胞的吞噬功能3间接触事件已经通过混合两种类型的细胞成像，然后连续扫描场的观点，找到适当的互动阶段偶然间接触。这些事件的随机性，使这个过程繁琐，它是通过这种方法难以观察到在细胞与细胞接触的早期或稍纵即逝的事件。这种方法需要发现细胞对接触的边缘上，并观察他们，直到他们完善他们的联系，或不。为了解决这些限制，我们使用光学诱捕作为一种非侵入性，非破坏性的，但快速和有效的方法定位在培养细胞。 在生物学研究中越来越多地利用光学陷阱，或光镊，捕捉和物理操纵细胞和其他微米大小的颗粒在三个层面 4 。首先观察到的辐射压力和应用光镊系统于1970年5，6，最初是用来控制在1987年7的生物标本。从那时起，光镊技术已经成熟到探头8-13多种生物现象。 我们描述的方法14，进步活细胞成像，通过与纺丝温度和湿度控制的磁盘共聚焦显微镜，提供精致的空间和时间在生理环境中病原微生物的控制，以方便与宿主细胞相互作用，确定由一个集成光学陷阱运营商。活，如白色念珠菌和曲霉 ，这可能会导致免疫功能低下15个人，16（ 例如艾滋病，化疗和器官移植患者）的潜在致命性，侵袭性感染，病原微生物光学被困采用非破坏性的激光强度和移动相邻的巨噬细胞，它可以吞噬病原体。高分辨率，传输光，荧光为基础的电影的建立能力，观察活细胞的吞噬功能的早期事件。为了证明在免疫学的广泛适用性，主要的T细胞也被困和操纵与抗CD3 涂层微球在体内形成的突触和突触形成的成像时间推移也获得。通过提供一个方法来施加罚款活病原体与免疫细胞的空间控制，细胞相互作用可以被捕获，以最小的扰动细胞荧光显微镜和先天免疫和适应性免疫的早期反应可以产生强大的洞察力。

Watch this Scientific Journal Video about Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging at JoVE.com

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

一种方法是描述单独选择，操作和使用光学陷阱，再加上一个旋转的磁盘显微镜图像活病原体。光陷阱提供了空间和时间的控制生物体和地方他们相邻的宿主细胞。荧光显微镜捕捉动态，以最小的扰动细胞间的相互作用。

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

use-an-optical-trap-for-study-host-pathogen-interactions-for-dynamic

Research

JoVE Journal

Immunology and Infection

12.0K 次观看.  Massachusetts General Hospital, Harvard Medical School. 一种方法是描述单独选择，操作和使用光学陷阱，再加上一个旋转的磁盘显微镜图像活病原体。光陷阱提供了空间和时间的控制生物体和地方他们相邻的宿主细胞。荧光显微镜捕捉动态，以最小的扰动细胞间的相互作用。

视频: Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

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

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

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

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

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

科研

免疫与感染

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

制备光学质量玻璃表面研究巨噬细胞融合

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...