这项工作是支持由美国国立卫生研究院授予P01人工智能068730（SNC，NS），美国国立卫生研究院授予CA016520 / TAPITMAT（NS），从Claneil基金会（NS）的私人资金，和卵巢孢子授予“气候变化框架公约”和美国宾夕法尼亚大学（ P50 CA83638）和福克斯蔡斯癌症中心的核心格兰特（P30 CA06927）（DCC）的。作者感谢的光/生物发光博士EJ Delikatny执导，安东尼秘密的宾夕法尼亚大学在美国宾夕法尼亚州癌症大学博士G. Danet - Desnoyers定向干细胞和异种核心的核心，优秀的技术援助中心培训SNC的原位注射技术，在美国宾夕法尼亚州/ OCRC大学Denada Dangaj协助手术。

1。细胞培养<ol><li>前原位注射，文化MOV1 吉细胞，DR26肿瘤所得在T175烧瓶，直到他们90％融合。计划用1至5亿个细胞，每次注射，这将需要1或2 T175烧瓶。 </li><li>注射当天，收获细胞，并确定使用血球细胞数。 </li><li>一旦细胞浓度已经确定，颗粒细胞离心5分钟，在室温下在300 X克。 </li><li>旋转，悬浮细胞，在10微升无菌PBS 1万，0.002 M EDTA </li></ol> 2。手术前<ol><li>手术前，填补了一个3/10cc胰岛素注射器1万MOV1 吉在10微升的PBS EDTA细胞。 </li><li>转让isofluorane麻醉鼠标加热垫。添加眼药膏，以防止眼部脱水。然后，立即插入一个鼻锥系统连接到一个异氟醚蒸发器，以提供整个手术的麻醉动物的头部。 </li><li>注射部位用酒精棉签消毒后，皮下注入酮洛芬，术前镇痛药5毫克/公斤，与3/10cc胰岛素注射器。 </li><li>使用快船，​​刮背的左边，从胸腰椎交界的尾部部分动物尾巴的基地。应用脱毛膏彻底清除头发。然后，取出用湿纸巾的过量。 </li><li>一旦头发已被删除，消毒用碘伏和酒精棉签剃光面积。然后，将手术切口区周围的悬垂。 </li></ol> 3。外科<ol><li>只需在手术前，调整至1.5％的异氟醚蒸发器水平。 </li><li>验证捏垫脚的动物是完全麻醉。 </li><li>接下来，找到皮肤下的脾脏。然后用手术剪，一个背外侧切口长1-2厘米，脾右上方。 </li><li>解剖后腹膜。将观察到小鼠卵巢周围的垫。用弯钳把握和暴露小鼠卵巢周围的脂肪垫。 </li><li>水合物的器官与一些滴无菌PBS。 </li><li>用弯钳把握，收回... ...位置... ...然后安全注射卵巢</li><li>虽然牢牢把握镊子卵巢，卵巢注入MOV1 吉肿瘤细胞的10微升。一个牢牢把握，防止液体返流或泄漏。 </li><li>注射后立即释放镊子施加的张力。卵巢穿孔自发地缩回并关闭。 </li><li>使用可吸收聚乙醇酸缝合针，关闭复古腹膜伤口。 </li><li>推出动物的鼻锥。 </li><li>拉伸皮肤和几滴组织粘合剂与密封背侧的伤口边缘。 </li><li>最后，口服抗生素管理100微升的动物。然后将它在笼子里，并恢复显示器。一个星期保持在饮水中的抗生素的动物。 </li></ol> 活体成像4。 <ol><li> MOV1 吉肿瘤细胞原位注射一个星期后， 在体内成像执行。开始转移isofluorane麻醉鼠标成像室。 </li><li>关闭异氟醚蒸发器的水平下降到2％。 </li><li>根据成像系统制造商的指示执行，在体内成像。在这个视频，Lumina的系统将被使用。 </li><li>形象，点击的生活图像软件桌面上的图标。然后，在IVIS采集控制面板，选择“初始化”。仪器设置类似的相机设置</li><li>在收购控制面板设置仪器的采集参数。荧光，检查“荧光”。点击照片框，以获取与每一个形象照片。 </li><li>接下来，设置为自动曝光时间。根据“像素合并或CCD分辨率”，“中等”。的F /停止或光圈，然后根据检查的值2。下一步，选择535激发滤光片和DsRed的排放过滤器。 </li><li>根据视野，形象一个单一的鼠标点击视图B。 </li><li>然后，点击获得的图像采集开始。 </li><li>一旦完成图像采集，使用感兴趣区域，或投资回报率，信号的工具来测量。测量图示上按一下释放的信号值和面积。 </li><li>最后，点击“保存”，保存在用户文件夹中的图像</li><li>已保存的图像后，停止异氟醚的管理和鼠标返回到笼子里。鼠标应该立即起床。 </li></ol> 5。代表性的成果使用此协议原位卵巢癌的生长， 在体内可以监控使用一种非侵入性的程序至少3周。 <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="图1" /> 图1。MOV1 吉细胞，PBS为阴性对照，原位注入卵巢非肿瘤易感小鼠（对动物和左边的动物，分别）。 体内成像2周后进行。 MOV1 吉在卵巢嫁接的细胞所产生的荧光发射测量，与阴性对照组小鼠相比。 <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="图2" /> 图2。MOV1 吉卵巢肿瘤冰冻切片染色与生物素标记的抗CD4单克隆抗体由民建联基板（深褐色）检测肿瘤浸润淋巴细胞（黑色箭头）。细胞counterstained甲基绿色可视化细胞的细胞核（蓝色）。 T细胞的形态，从不同的肿瘤细胞（红色箭头）。幻灯片的显示放大40倍。

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手术和原位注射卵巢囊原位注射要求的培训和精度。因此<ol><li>可怜的手术经验，实践与尸体第一。 </li><li>使用优先multiparous女性（一个或两个升）作为他们发展，随着时间的推移有利于注射和提高生存与未产妇女性更大卵巢。 </li><li>由于小鼠卵巢囊体积小，使用的最小可用针头大小的大力鼓励。 </li></ol> 在活体成像<ol><li>始终使用荧光，如1.5毫升...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM-GLUTAMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10564-011</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14040</td>
<td>&nbsp;</td>
<tr>
<td>Versene</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-711E</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Deltaphase</td>
<td>39 DP</td>
<td>&nbsp;</td>
<tr>
<td>Povidone pads</td>
<td>&nbsp;</td>
<td>Dynarex</td>
<td>1108</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>06-669-62</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tears ointment </td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>17845-153</td>
<td>&nbsp;</td>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Fort Dodge laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3cc/insulin syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309301</td>
<td>&nbsp;</td>
<tr>
<td>Polyg Polyglycolic Acid suture/needle (3/8 19mm)</td>
<td>&nbsp;</td>
<td>Syneture</td>
<td>9612-31</td>
<td>&nbsp;</td>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>Vetbond</td>
<td>3M</td>
<td>&nbsp;</td>
<tr>
<td>Vet Bactrim/ oral suspension</td>
<td>&nbsp;</td>
<td>Hi-tech Pharmacal</td>
<td>840823</td>
<td>&nbsp;</td>
<tr>
<td>IVIS-Lumina</td>
<td>&nbsp;</td>
<td>Caliper lifesciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isofluorane</td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>J108013</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum, Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437036</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>TurboFP635 mammalian vector</td>
<td>&nbsp;</td>
<td>Evrogen</td>
<td>FP721</td>
<td>&nbsp;</td>
<tr>
<td>T175 flasks</td>
<td>&nbsp;</td>
<td>cellstar</td>
<td>660-190</td>
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an orthotopic model of serous ovarian cancer in immunocompetent mice for in vivo tumor imaging and monitoring of tumor immune responses

背景：卵巢癌是一般诊断为晚期的情况下/病死率高，因此仍是最致命之间的美国妇女1,2,3的所有妇科恶性肿瘤。浆液性肿瘤卵巢癌的最普遍的形式和4,5 TG - MISIIR标签转基因是唯一的，自发地发展这种类型的肿瘤的小鼠模型。 TG - MISIIR标签小鼠表达SV40病毒转化地区的苗勒氏管抑制物质II型受体（MISIIR） 第 6基因启动子的控制之下。额外的转基因株系已经确定，表达转基因SV40的标签，但不发展卵巢肿瘤。非肿瘤易感小鼠C57BL / 6小鼠表现出典型的寿命和肥沃。这些小鼠可用于隔离TG - MISIIR - TAG - DR26小鼠肿瘤细胞同源移植。 目的：虽然肿瘤显像7，小动物活体深部肿瘤的早期发现是具有挑战性的。为了使浆液性卵巢癌的免疫完整的动物模型的临床前研究，我们描述了一个这种类型的卵巢癌，在活体成像，肿瘤微环境与肿瘤的免疫反应的研究许可证的同源小鼠模型。 方法：我们首先来自收获一种自发的在26周岁的DR26 TG - MISIIR标签的女性卵巢肿瘤标记+鼠标癌症细胞株（MOV1 ）。然后，我们稳定转导MOV1 TurboFP635的慢病毒哺乳动物载体，编码Katushka， 海葵Entacmaea quadricolor红色荧光蛋白635分之588 纳米 8,9,10极大激发/发射远红外突变细胞。我们原位植入MOV1 吉非肿瘤容易发生TG - MISIIR标签女性小鼠卵巢11,12,13,14。其次是在体内光学成像和肿瘤微环境是由免疫组织化学分析的肿瘤进展。 结果：原位植入MOV1 吉细胞浆液性卵巢肿瘤。 MOV1 吉肿瘤可以在活体成像的可视化由植入后3周（图1），并与白细胞浸润，15（图2）在人类卵巢癌的观察。 结论：我们描述了一个卵巢癌原位模型， 适合在高pH值的稳定，并在深部组织Katushka耐光由于早期肿瘤的体内成像。我们建议使用这种新颖的浆液性卵巢癌的同源模型，在活体成像的研究和监测肿瘤的免疫反应和免疫疗法。

Watch this Scientific Journal Video about An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses at JoVE.com

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

研究<em&gt;在体内</em&gt;肿瘤的生长和肿瘤微环境中，我们用在免疫动物的同源和原位卵巢癌的小鼠模型。我们转Katushka荧光蛋白（MOV1一只老鼠的肿瘤细胞株（MOV1）<sup&gt; KAT</sup&gt;），我们在这里展示其在卵巢原位移植和<em&gt;在体内</em&gt;成像。

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

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Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

18.1K 次观看.  University of Pennsylvania-School of Medicine. 研究在体内肿瘤的生长和肿瘤微环境中，我们用在免疫动物的同源和原位卵巢癌的小鼠模型。我们转Katushka荧光蛋白（MOV1一只老鼠的肿瘤细胞株（MOV1） KAT），我们在这里展示其在卵巢原位移植和在体内成像。

视频: An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

科研

免疫与感染

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

小鼠中人类胸膜皮质瘤的PET/CT的植入与监测

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

肿瘤微环境下免疫细胞种群的可视化、定量和映射

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

用于剖析体外肿瘤微环境中免疫反应的微流控共培养模型

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.