This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) M&#252;nster (SEED 03/12, SB), Deutsche Forschungsgemeinschaft (SFB TR128, TP B6 to S.G.M. ME3283/2-1 to S.G.M.) and by Innovative Medizinische Forschung, M&#252;nster (I-BI111316, SB and SGM).

使用小鼠所有实验应根据各自的机构动物护理和使用委员会的指导方针进行。 1.一般注释小鼠实验<ol><li>保持小鼠无病原体的条件下，使它们获得食物和水随意。 注：由于免疫模式可以与年龄和性别的不同是非常重要的使用年龄和性别匹配的小鼠实验组。 </li></ol> 2.制备和活化的OVA特异性CD8 + T细胞（OT-I） <ol><li>为准备脑片之前描述如下5天执行OT-I T细胞的刺激。 注：5×10 5活化的效应的CD8 + OT-I的T细胞（CD8 + T细胞的转基因小鼠识别只有OVA 257-264肽的MHC-I类分子的上下文中），需要每片的浓度为5× 10 5被选为实验同盟作为最佳的一种以有利于一个良好的细胞-细胞相互作用，一旦这些细胞开始迁移到切片，并在同一时间，细胞的量不太高，以诱发过度反应，允许单细胞计数8,18。 </li><li>备的OT-I的T细胞培养的培养基。补充500毫升DMEM，5％胎牛血清（FCS），10mM的HEPES，2mM的L-谷氨酰胺，50μM2-巯基乙醇，1％非必需氨基酸和25微克/毫升庆大霉素。储存在4℃直至使用的介质。 </li><li>除去的OT-I转基因小鼠的脾按照参考16。他们转移到准备好的媒介。 </li><li>通过一个70微米的过滤器和离心机悬浮糖化脾脏，在300×g离心5分钟，4℃产生单细胞悬浮液。 </li><li>孵育细胞悬浮液用5ml铵氯化物-钾（ACK），缓冲液（150mM的氯化铵 ，10mM的KHCO 3，0.1mM的EDTA，pH7.3）中进行5分钟，以裂解红细胞。 </li><li>停止incubati上用10毫升培养基并离心再次如上所述。 </li><li>稀释细胞悬浮在培养基中，并计算数字。板的细胞以3×10 7个细胞的密度/孔在12孔板和素它们温育以OVA 257 - 264（SIINFEKL;为1nM）和白细胞介素-2（IL-2）（500国际单位/毫升），用于5天。 </li><li> 4天后，/毫升再次添加的IL-2在500国际单位的浓度。 </li><li>随后，以刺激，纯化的OT-I的T细胞，从细胞悬浮液通过使用负选择基于小鼠的CD8 + T细胞分离试剂盒按照生产商的说明。纯化是通过用抗CD4，细胞CD11b，CD11c的，CD19，CD45R（B220），CD49b（DX5），CD105，MHC II类，泰尔-119，和TCRγ的鸡尾酒基于所有非CD8 +细胞的耗竭/δ其中通过使用磁列，用于纯化的CD8 + T细胞的洗脱然后丢弃。 注意：在此程序，可以关键步骤再指示由所述生产：重要的是，该反应仅涉及单个细胞，因为团块的存在可能在洗脱步骤阻止柱;小区添加Byotin鸡尾酒之前计数是用于样品的纯度程度重要并且过程应在冰上进行，以尽量减少非特异性抗体绑定。 </li></ol> 3.准备急性脑切片和共培养与OT-I T细胞<ol><li>现有急性脑的制备切片19，制备用200mM的蔗糖，20毫摩尔PIPES，2.5mM的氯化钾，1.25毫的NaH 2 PO 4，10mM的硫酸镁 ，0.5 氯化钙和10mM葡萄糖placedine冰冷的生理盐水溶液和人工脑脊液（ACSF）通过使用125 mM氯化钠，2.5氯化钾，1.25的NaH 2 PO 4，24 mM的碳酸氢钠 ，2毫硫酸镁 ，2毫的CaCl 2，10mM的葡萄糖。由全光照pH调节各溶液，以7.35克NaOH。前调节pH值ACSF的，溶液必须通入95％O 2和5％ 的 CO 2的混合物。 </li><li>预冷却的解决方案。 </li><li>麻醉8 - 10周大的小鼠（ 例如，C57BL / 6对照或转基因的ODC-OVA小鼠17与吸入用5.0％异氟烷，5.0％氟烷或通过IP注射100mg / kg的氯胺酮和10mg / kg体重甲苯噻嗪等待麻醉，并使用脚趾捏来评估麻醉水平和斩首一次他们。安乐死小鼠按照机构动物护理和使用委员会标准安乐死。 </li><li>把鼠标腹位置。消毒，切头皮矢状。打开颅骨矢，迅速用瓢的帮助下取出脑并用胶水将其固定到一个的vibratome的板块。 </li><li>填充板与制备placedine冰冷的生理盐水溶液。 </li><li>削减300微米冠状切片用的vibratome。 注意：这个程序是知道n至得到适合于至少8小时19-21的时间间隔内功能性细胞的研究活完整的组织标本。事实上，在这一段时间内，6小时后已经开始，该制剂显示质量降低的，即改变像动作电位的产生和传播的基本和功能性质。 </li><li>切片后，立即传送一个片成12孔板填充ACSF的每个孔中。 </li><li>每片加小心5×10 5的OT-I的T细胞。 </li><li>孵育切片在培养箱多达8小时（37℃，5％的CO 2）。 </li><li>潜伏期，收获切片后嵌入他们通过使用十月复合组织-TEK，并冻结他们在液氮。存储它们在-20℃用于进一步的组织学研究，以例如评估神经结构（ 例如，使用抗MAPII或抗突触抗体）或细胞凋亡（ 例如，使用抗Caspase-3的抗体和抗的NeuN反体）按照标准方法。 </li></ol>

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The authors declare that they have no competing financial interests.

不同的动物模型已经描述在过去的几十年来解决的炎性脱髓鞘疾病如MS的病理特征。 体内小鼠和大鼠模型被广泛用于模拟疾病的病理生理特征，即，脱髓鞘和髓鞘再生过程的后果分析和炎症和神经退行性疾病的混合体发作。然而，只有一个离体方法允许解剖确切基本机制。 也是急性脑切片制备一个广泛分布的模型，并可以被认为是可靠的和可复制的。在与隔离?...

Death of oligodendrocytes and destruction of the myelin sheath accompanied by loss of neurons and axons are typical pathological findings in grey matter lesions in individuals suffering from multiple sclerosis (MS)1,2. Cortical lesions can be divided so far in three different subtypes2: subpial, intracortical and leukocortical lesions. In comparison to white matter plaques, infiltrates are characterized by a predominance of CD8+ T-cells, suggesting their possible decisive role in grey matter inflammation3. Furthermore, oligoclonal expansions in blood, cerebrospinal fluid (CSF) and within inflammatory lesions can be found for CD8+ T-cells themselves4-6. 
In line with this, it is assumed that CD8+ T-cells may be specific for different myelin proteins7,8. Indeed, CD8+ T-cells are found near oligodendrocytes and myelin sheaths9,10 that show MHC I expression11 and might therefore be responsible for the loss of the myelin sheath. This process is often seen together with extensive &#8216;&#8216;collateral&#8217;&#8217; neuronal and axonal damage within the central nervous system (CNS) grey matter1,2. In fact, direct and indirect death of oligodendrocytes and neurons is induced by CD8+ T-cells via two different mechanism: (i) cell membrane swelling and rupture due to the formation of cytotoxic granules following the release of perforins and granzymes and (ii) ligation to the Fas receptors or exposition of FasL on their surface8,12,13.
Different types of animal models are established to study the underlying mechanism of the mentioned processes. In this respect, primed CD8+ T-cells specific for autoantigens with induced expression in CNS glial cells, like oligodendrocytes or astrocytes, can be adoptively transferred to analyse &#8216;&#8216;collateral&#8217;&#8217; neuronal and axonal death in grey matter subsequently14,15. To perform such in vivo experiments is a big help to mimic some pathophysiological aspects of MS, however, this approach is not suited to resolve the underlying mechanism and kinetics of axonal damage and neuronal apoptosis.
To overcome these restrictions, an ex vivo approach was established to study the mechanisms and time course of neuronal cell death following a oligondendrocytes-directed CD8+ T-cell attack. Since only oligodendrocytes and therefore myelin sheath production should be targeted by immune cells, MHC class-I-restricted, ovalbumin (OVA)-reactive OT-I Tcells are used16. These cells are subsequently transferred into brain slices obtained from mice selectively expressing OVA in oligodendrocytes (ODC-OVA mice)17.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:264px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">12-Well-plate</td>
			<td style="width:187px;">Corning</td>
			<td style="width:119px;">3513</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Mercaptoethanol</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">31350-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Methylbutan</td>
			<td style="width:187px;">Roth</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">70 &#181;m strainer</td>
			<td style="width:187px;">Falcon</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CaCl2</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.02382.0500</td>
			<td>calcium chloride</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CD8+-isolation kit</td>
			<td style="width:187px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-859</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">D(+)-glukose</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.08337.1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DMEM</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td>warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">EDTA</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">E5134</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">FCS</td>
			<td style="width:187px;">PAA Laboratories</td>
			<td style="width:119px;">A15-151</td>
			<td>fetal calve serum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">gentamicin</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HEPES 1M</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">15630-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">IL-2</td>
			<td style="width:187px;">Peprotech</td>
			<td style="width:119px;">212-12</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Isofluran</td>
			<td style="width:187px;">Abbott</td>
			<td style="width:119px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">KCl</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.04933.0500</td>
			<td>potassium chloride</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">KHCO3</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">P9144</td>
			<td>potassium hydrogen carbonate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">L-Glutamine</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MgSO4</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.05886.0500</td>
			<td>magnesium sulfate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaCl</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">31434</td>
			<td>sodium chloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaH2PO4 * H2O</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.06346.0500</td>
			<td>sodium hydrogen phosphate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaHCO3</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.06329.0500</td>
			<td>sodium hydrogen carbonate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaOH</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.09137.1000</td>
			<td>sodium hydroxide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NH4Cl</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">213330</td>
			<td>ammonium chloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Non Essential Amino Acid</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">OVA (257-264)</td>
			<td style="width:187px;">Genscript</td>
			<td style="width:119px;">RP10611</td>
			<td>ovalbumin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PIPES</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">P6757</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Sukrose</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.07687.1000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Tissue-Tek OCT</td>
			<td style="width:187px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo model of an oligodendrocyte-directed t-cell attack in acute brain slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

脑切片与少突胶质细胞定向的CD8 + T细胞的培养后，少突胶质细胞以及神经元凋亡（ 图2A和图1C，分别）。 （ 例如，胱天蛋白酶-3，TUNEL）可以最早3小时孵育后检测细胞凋亡的组织学迹象。潜伏期不应该长于8小时，以保证在制备和可再现的结果的一个良好的品质。细胞凋亡，可以发现全国各地有优势的领域髓切片。片的结构的完整性和凋亡神经元的示例性组织染色中描?...

Watch this Scientific Journal Video about An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices at JoVE.com

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

an-ex-vivo-model-an-oligodendrocyte-directed-t-cell-attack-acute

Research

JoVE Journal

Immunology and Infection

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

6.8K 次观看.  University of Münster. To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

视频: An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

科研

免疫与感染

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号：对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...