这项研究得到了国防医学院的资助以及日本卫生、劳动和福利省为研究棘手疾病而提供的健康和劳动科学研究补助金的支持。

实验方案经国防医学院动物研究委员会批准（第16058号）。这些动物被保存在标准实验室周（CLEA日本公司，东京，日本）。实验室动物按照国家卫生研究院的指导方针进行治疗。
1. 淋巴细胞的收集和分离
注:由于淋巴细胞必须是新鲜的，不能储存，因此每次实验都必须从大鼠那里收集淋巴细胞。此外，肠道热带淋巴细胞必须直接从它们循环的胸管中收集。所有程序预计将在 6 小时内完成，所有仪器、手套和表面均清洁。为了使动物保持生理稳定状态，实验中使用的所有盐水和其他液体都应保持温暖。
<ol><li>将大麻管浸入热水（约80-90°C）后，形成一个像肝素曲线（半径约5毫米）一样的制罐管（直径1毫米），并提前几个小时用胶带将其固定在塑料板上。</li>
	<li>麻醉雄性威斯塔大鼠（8-12周），腹腔内注射米达佐兰（2毫克/千克）、多米托（0.15毫克/千克）和贝图尔帕尔（2.5毫克/千克）的混合物，并用剪发器剪下腹部的体毛。剃须后牢固地取出头发，使头发不会进入腹腔。</li>
	<li>在确认手术所需的麻醉深度稳定后，通过脚趾捏捏，从中线到左下部区域水平用手术剪刀切口。小心不要通过观察光下的血管来损坏腹膜内的血管。</li>
	<li>用湿纱布包裹胃部，将胃轻轻地移到身体的右侧，以揭示反胃器官。使用手术剪刀在脊柱直立肌肉和脂肪组织之间插入一个缺口，并用手指将它们直接分离。</li>
	<li>小心地剥去胸管周围的结缔组织。多余的结缔组织会随着年龄的增长而增加。从隔膜的左十字下方露出胸管，直到可见胸管的20毫米长度。使用精密钳子或湿棉棉签轻轻地将胸管和主动脉之间的粘附物分开。<ol>
<li>仔细执行这个过程，因为一些大鼠有动脉从腹部动脉分支穿过胸管或短胸管由于淋巴丛（小胸管像蜘蛛网一样蔓延）的较高位置。避免不必要的接触胸管，以避免诱发水肿。</li>
	</ol>
</li>
	<li>在隔膜左十字下方的胸管上用绳子（3-0丝）涂上连结。胸腔胸管变得分散。</li>
	<li>通过刺穿手术剪刀的尖端，通过发夹弯曲的针管，并在某一点上将管子放到 iliopsoas 肌肉上，在腹部壁上打一个洞（直径 5 毫米）。用含有10U/mL肝素的普通盐水填充罐装管。</li>
	<li>在松开的胸管下放置一根绳子（3-0根丝绸）后，用罐管的锋利边缘刺穿胸管，将其朝向尾巴约5毫米，用罐装管将胸管固定。</li>
	<li>为了提供盐水以防止脱水，使用精密钳子在胃的蚂蚁前壁上创建一个孔（直径3毫米），并通过火焰环将硅管（直径2毫米）传给十二指肠。缝合伤口并维持波尔曼笼中的动物后，开始以3mL/h的流动速度从硅管中将含糖盐水注入每只老鼠的十二指肠。用纸巾盖住整个笼子，让它保暖。</li>
	<li>将制冷管固定在冰上含有6 U/mL肝素、10%胎儿牛血清和RPMI 1640介质（pH 7.4;见材料表）的50mL圆锥管盖的孔中，并在冰上收集胸管淋巴细胞（TDLs）。小心不要将管子的尖端接触到圆锥管的壁上，以防止由于管内纤维蛋白血块的形成而导致的罐管闭塞。淋巴液（约20 mL），包括约1.0 x 108~109淋巴细胞可以在6小时内获得。为了在完全麻醉下获得淋巴细胞，对麻醉平面进行监测，并适当观察淋巴流量。同样的麻醉剂在老鼠被放入鲍尔曼的笼子后大约3小时以同样的剂量添加，以连续接受6小时的深度麻醉。</li>
</ol>2. 淋巴细胞标签与卡盒氟雷西汀二乙酸苏奇米迪尔酯 （CFDSE）
<ol><li>溶解 CFDSE （材料表） 在二甲基硫化物 （DMSO） 到 15.6 mM （500μg 的 CFDSE 溶解在 60 μL 的 DMSO）。</li>
	<li>在RPMI 1640的50 mL中孵化淋巴细胞（1 x 108~109），在37°C下孵化50微升的CFDSE溶液，30分钟，如前10次所述。</li>
</ol>3. 微血管研究实验设置
<ol><li>麻醉接受者大鼠（8-12周）与腹膜注射的混合物米达佐兰（2毫克/公斤），多米托（0.15毫克/公斤）和贝图尔帕尔（2.5毫克/公斤），并通过脚趾捏确认麻醉的深度。在梳理毛皮之前将腹部弄湿，以尽量减少头发污染。然后通过中线切口打开接受者威斯特大鼠的腹部。</li>
	<li>将鼠放在便携式不锈钢板上（约 120 x 300 mm），中心周围有一个长方形孔，上面覆盖着玻璃滑梯（24 x 50 mm）。选择约10厘米的伊利尔段，包括PP进行观察。</li>
	<li>保持肠道尽可能温暖和潮湿与磷酸盐缓冲盐水（PBS）加热到37°C。 浸泡纱布，用于覆盖小肠与PBS。</li>
	<li>同时用2%的异氟化物进行连续麻醉（见材料表），将滑梯放在显微镜的舞台上，并选择合适的区域来观察一些高清病毒穿过血清的PP中的微循环。PP 的大小范围;较大的适合使用 CLSM 观察微循环。小肠在有些地方不直:一个至少2厘米长的直段，没有任何张力是适合观察。</li>
	<li>覆盖相邻的肠道部分，用浸泡在PBS的吸水棉中浸泡。将肠道部分放置在两个轧制的棉球之间，并尽可能将其从大鼠体内放置，以防止它被老鼠的心跳和呼吸振动。</li>
	<li>使用 1 mL 注射器，缓慢地将 （超过 1 分钟） CFSE 标记的 TDLs （1 x 108 细胞） 注射到受体大鼠的血管中。快速静脉注射可能会影响系统循环。</li>
</ol>4. 淋巴细胞的微循环
<ol><li>使用 CLSM 持续监控 PP 微血管中的 TDL，并每隔 30 分钟在计算机上使用延时摄影记录 3 小时。从塞罗萨到PP的 HEV 深度约为 25μm，使对频闪和毛细血管的观测深度达到 30μm。</li>
	<li>将得克萨斯红-德克斯特兰（25毫克/千克）注射到每个受体大鼠的血管中，以弄脏血液，将霍赫斯特33342（5毫克/千克）染色细胞核。</li>
	<li>（可选）要量化淋巴细胞动力学，将粘附于 HEV 的淋巴细胞定义为"粘合淋巴细胞"，将从 HEV 迁移到频闪的淋巴细胞定义为"迁移淋巴细胞"。然后计算每个视野（约0.3毫米2）迁移的平均百分比（迁移淋巴细胞/粘合淋巴细胞+迁移淋巴细胞）。</li>
</ol>

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作者没有什么可透露的。

在这里，我们描述了收集天真的肠道热带淋巴细胞和观察它们在大鼠PPS中的迁移的协议。这些程序可以揭示淋巴细胞在PP的微血管中如何移动，并使得在正常或用药条件下对它们的动态进行视觉比较成为可能。直接观察这些动态有很多优点，以获得一些药物的免疫修饰的线索，虽然观察期仅限于几个小时。
我们在方法中提到了一些提示。其中，最微妙的程序之一是操作员必须...

佩耶的斑块（PPs）由小肠的拉米纳柱状物中的数百个淋巴状毛囊组成。PPs分为卵泡、间曲区域和位于毛囊下部的细菌中心，其中淋巴细胞受到抗原表现的刺激。没有发泄淋巴血管，抗原通过上皮细胞层从肠道流明侵入拉米纳丙酸。覆盖淋巴卵泡的上皮区域称为卵泡相关上皮，其中专门穿插M细胞吸收粘膜抗原。M细胞从发光侧摄取抗原，然后由支气管细胞捕获抗原，并呈现给通过高内皮静脉（HEV）1的内皮流入PP的天真淋巴细胞。PPs在肠道免疫中起着重要作用，与炎症的早期阶段有关。许多分子相互作用涉及淋巴细胞进入二级淋巴器官（SLOs），包括粘附分子、化疗因子2、3和磷酸-1-磷酸盐4:因此，有许多预期的治疗目标。因此，观察PP内淋巴细胞动力学使我们能够瞥见炎症的早期阶段，并检查几种有希望的药物的用处。
这里的方法侧重于PP中淋巴细胞的迁移，其中包括几个程序（可以进入胸管5， 收集淋巴细胞和注射到收集淋巴细胞后的长期观察）。由于这些程序很复杂，很难确切地看到以前的报告中每个程序是如何执行的，因此我们在这里提到了一些实现成功观察的技巧。例如，将管子插入胸管非常困难，制精的初始成功率不到50%。然而，我们改进了方法，成功率超过80%。我们在此手稿中提到了一些其他提示，这些提示对于成功观测以实现对淋巴细胞在若干条件下的跨内皮迁移进行定量评估是必要的。
在以前的报告中，很难理解随着时间的推移的三维变化，如静脉注射印度墨水染色PP6的血管结构，或显微镜是单焦7。近年来，利用凯德小鼠等一些光可转换荧光蛋白转基因动物的观察方法，澄清了体内的系统细胞运动。另一项研究澄清了CD69从PP9独立关闭淋巴细胞出口。由于其高分析能力，我们使用共分体激光扫描显微镜 （CLSM）。现在，我们可以很容易地获得高分辨率图像，并用它来分析淋巴细胞动力学。
在这份报告中，我们演示了一系列评估PP淋巴细胞迁移的方法。首先，我们展示了收集淋巴细胞的胸管罐装的精制方法。其次，我们改进了观察方法，在微观观测下尽可能保持客观器官，从而能够获得3小时的高质量图像。第三，我们量化淋巴细胞迁移的细胞运动，以评估某些药物的影响。这些修改后的协议将有助于开展粘膜免疫学评估。

<table><tbody><tr><td>A1R+</td><td>Nikon</td><td></td><td>Comfocal Laser Scanning Microscopy</td></tr><tr><td>Carboxyfluorescein diacetate succinimidyl ester</td><td>Thermo Fisher Scientific</td><td>C1157</td><td></td></tr><tr><td>Hoechest 33342</td><td>Thermo Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>Isoflurane</td><td>Wako Pure Chemical Industries</td><td>099-06571</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>GIBCO</td><td>11875093</td><td></td></tr><tr><td>Texas Red&amp;ndash;dextran</td><td>Thermo Fisher Scientific</td><td>D1863</td><td></td></tr></tbody></table>

microscopic observation of lymphocyte dynamics in rat peyer's patches

天真的淋巴细胞在生理条件下从血液循环到淋巴组织，被公认为肠道免疫中的一个重要现象。二级淋巴细胞器官的频谱，如Peyer的斑块（PPs）或中性淋巴结，是天真淋巴细胞感知抗原的地方。天真的淋巴细胞通过血液循环，以达到高内皮静脉，进入PP的入口。据估计，一些免疫调节器会影响淋巴细胞迁移，但对微循环动力学的精确评估非常困难，建立体内淋巴细胞迁移观测方法有助于澄清精确机制。我们改进了从淋巴管中收集淋巴细胞的方法，并观察了大鼠PP中肠道淋巴细胞的详细动态。我们选择了折叠式激光扫描显微镜来观察体内的老鼠PP，并使用延时摄影进行记录。我们现在可以获得清晰的图像，有助于淋巴细胞动力学的分析。

从淋巴中收集淋巴细胞 要为大鼠准备胸管罐头，如 图1 所示，在紧张的胸管中切口，然后将大鼠维持在波尔曼的笼子中，如 图2所示。
当淋巴细胞收集良好时，我们可以获得约20 mL/6 h淋巴液，含有约107~108/mL淋巴细胞。在 TDL 中，70% 的快速 CD4，其中约 90% 的细胞表示 CD62L，这意味着 TDL 的主要组成（+6...

Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

在这里，我们描述了一个精确的方法，收集胸管淋巴细胞和观察肠道热带淋巴细胞在老鼠Peyer的补丁的迁移3小时使用延时摄影。这项技术可以澄清淋巴细胞的动态在炎症条件下是如何受到影响的。

鼠佩耶斑块中淋巴细胞动力学的微观观察

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

microscopic-observation-of-lymphocyte-dynamics-in-rat-peyer-s-patches

Universidad de Cartagena UDEC

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JoVE Journal

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3.9K 次观看.  National Defense Medical College. 在这里，我们描述了一个精确的方法，收集胸管淋巴细胞和观察肠道热带淋巴细胞在老鼠Peyer的补丁的迁移3小时使用延时摄影。这项技术可以澄清淋巴细胞的动态在炎症条件下是如何受到影响的。

视频: Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

从小鼠的补丁中提取体液免疫的关键参与者: 先进和优化的淋巴细胞分离协议

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

癌症研究

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 ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

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

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

牵引力显微镜研究B淋巴细胞激活

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

生物工程

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

小鼠皮肤和肿瘤淋巴血管白细胞出口的定量

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

生物学

Isolation of Lymphocytes from a Mouse Peyer&#39;s Patch

Source: Yazicioglu, Y. F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58490">Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s Patches.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the isolation of lymphocytes from a harvested mouse Peyer&#39;s patch. The isolated lymphocytes can be used for further analysis.

从小鼠 Peyer 贴剂中分离淋巴细胞

Source: Yazicioglu, Y. F., et al. Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s ...