爱丽丝高根和王娇静，其优秀的技术支持，智力投入，并提前将这些研究在道义上的支持，我们非常感激。这些研究是由美国国立卫生研究院资助AI036532支持GAH。

1。致命的辐射<ol><li>放置最多10个受体小鼠成microisolator笼与要使用的照射装置兼容。 </li><li>照射总剂量（总剂量为10 cGy的BALB.B收件人）在同等剂量总结。二照射3小时后第一个。注射液发生后，最终照射4-6小时之间。无论是铯-137源或RS2，000，辐照的照射小鼠。 </li><li>酸化水之后的第二次辐射剂量，储存小鼠在microisolator笼，直到移植的时间。 </li></ol> 2。脾细胞的制备<ol><li>根据机构的指导方针，实施安乐死一个供体小鼠。每一个捐赠者通常会产生75x10 6 - 125x10 6脾细胞。使用CD8纯化试剂盒，将每只小鼠一般产生6×10 6 - 12×10 6 CD8 + T细胞。 </li><li>先垂直2厘米切口2厘米的中线直接取出脾脏下的肋骨。通过毛，皮，内脏膜切割。 </li><li>取出脾脏和创建将在40微升目筛培养皿中，用5％FBS的1640 RPMI脾单细胞悬液。使用注射器柱塞掰开脾脏，直到整个脾已经通过目筛网。 </li><li>重复此过程，每个WT和L2G85.B6捐助的。收集所有的WT单细胞悬浮液1-50毫升锥形管，收集所有的L2G85.B6在一个单独的50毫升锥形管的单细胞悬液。 </li><li>离心细胞在1,200 rpm离心10分钟，在4℃下粒料1640的RPMI重悬了5％FBS和计数细胞。 </li></ol> 3。骨髓准备<ol><li>去除皮肤的供体小鼠的后肢。 </li><li>小心地剪去股骨和胫骨/腓骨尽可能多的肌肉组织。 </li><li>删除后肢通过切割股骨远在髋关节。切掉后的爪子正下方胫骨/腓骨路口的。截骨应使用坚固的剪刀。 </li><li>小心地取出所有剩余的肌肉组织。切断腓骨，相对较少的骨髓中发现腓骨和不值得的努力。 </li><li>冷1640的RPMI 5％FBS和重复过程中与所述第二后肢后肢放置。每只小鼠20×10 6 - 40×10 6骨髓细胞之间产生。 </li></ol> 4。骨髓清除<ol><li>删除一个后肢从大的陪替氏培养皿中的媒体和地方，用少量的冷媒体（〜1毫升）。 </li><li>剪去膝关节。使用注射器（体积&gt; 5毫升），胫骨和抑制注射器插入皮下针，直到所有的红色物质是从内部胫骨。股骨重复此过程。丢弃剩下的骨头没有骨髓。第二后肢重复此过程。 </li><li>创建到大培养皿中，并使用注射器的柱塞和40μL移液含有骨髓单细胞悬液目筛。移取单细胞悬液50毫升锥形管，并保持在冰上。 </li></ol> 5。 CD3耗尽有很多种方法，以消耗CD3 +细胞从骨髓。我们的实验室使用的一个工具包，由德国美天旎生物技术公司（CD3-生物素 - 130-093-021）。消耗CD3 +细胞从骨髓制造商的协议。今后将被称为Miltenyi公司试剂盒的缓冲液MACS缓冲液（2毫摩尔EDTA，0.5％BSA的PBS，pH值7.2）。 <ol><li>洗涤细胞，以1200rpm离心分离的脾细胞和骨髓细胞的10分钟，在4℃，并计数细胞。 </li><li>删除ALL上清。的重悬骨髓细胞100μL，每10万骨髓细胞的MACS缓冲。 </li><li>与CD3消耗继续使用制造商的协议。 </li><li>洗净CD3贫乏的骨髓细胞在无菌PBS中的3倍。计数CD3耗尽骨髓细胞，并重新悬浮于适当的体积，注入10 7个细胞。 </li></ol>有很多种方法来净化从L2G85.B6小鼠的CD8 T细胞。此外，有几种方法来耗尽CD8 T细胞从WT脾细胞献血者。我们的实验室使用的试剂盒由德国美天旎生物技术公司（CD8消耗 - 130-049-401，CD8净化 - 130-095-236）。 <ol><li>在90μLMACS缓冲液每10 7细胞，重悬WT颗粒。根据制造商的协议（CD8消耗 - 130-049-401）继续CD8 T细胞耗竭。 </li><li>在40μl的MACS缓冲器，每10 7个细胞重悬于L2G85.B6颗粒。继续CD8 T细胞纯化，根据制造商的协议（CD8净化 - 130-095-236）。 </li><li>每个细胞计数人口，每个人口和洗3次，用无菌PBS。重悬在每个人口适当体积18×10 6 WT（CD8贫）脾细胞和2×10 6 L2G85.B6纯化的CD8 T细胞注入。 </li></ol>7。注射剂<ol><li>合并10 7 CD3耗尽的骨髓细胞，18×10 6 WT（CD8贫）脾细胞，2×10 6 L2G85.B6纯化的CD8 T细胞的微量离心管中。洗净，用无菌PBS重悬在300微升。 </li><li>到受体小鼠尾静脉注射细胞制备。应使用28号针头注射。 </li><li>将老鼠与酸化水microisolator笼。日常使用的得分分数小鼠移植物抗宿主病（GVHD）评分系统由库克等人 1。 </li></ol> 8。生物光学成像<ol><li>六天移植后受体小鼠，注射4毫克D-荧光素。允许5分钟虫荧光素与荧光素酶反应。 </li><li> 5分钟，小分级，麻醉小鼠中的生物发光成像仪和图像收件人的异氟醚室。这将创建一个高分辨率的图像，同时收集尽可能多的事件可能。 </li><li>分析数据使用活图片的软件。伪彩色图像的规模可以改变，以产生最好的结果。然而，这是必须的，整个实验中使用相同的刻度。 </li><li>可以创建，使用活体成像软件和发射的光通过计算（光子/秒），被发射的每个关心区域的磁通，可以量化的感兴趣区域。 </li></ol> 9。代表性的成果约7〜10天移植后，老鼠开始显示，移植物抗宿主病的临床症状。小鼠出现，邋遢由于缺乏疏导。收件人也将开始减肥移植后7-10天之间。将保持相对正常，直到大约每天12-14移植后受体小鼠的活动和姿势的。累积移植物抗宿主病（GVHD）的分数将稳步增长，通过移植后第2-3周（ 图1A）。病程是相当变数之间的小鼠，但是，收件人应均匀屈从于移植物抗宿主30-40ð的AYS移植后（ 图1B）。 图2显示了成像的6天移植后受体小鼠。伪彩色刻度显示不同的发射光的整个身体所发出的光的强度在脾脏和肠道的最高。 CD8 T细胞堆积在肠道中与以往的研究结果6是一致的。受体小鼠中，可以放回microisolator笼在稍后的时间点进行成像或安乐死的体外成像。 <table border="1"><tbody><tr><td> 标准 </td><td> 0级 </td><td> 1级 </td><td> 2级 </td></tr><tr><td> 减肥（wkly.） </td><td> &lt;10％ </td><td> &gt; 10％ - &lt;25％ </td><td> &gt; 25％的</td></tr><tr><td> 姿势 </td><td>正常</td><td> ，拱起只有在休息</td><td>几个拱起，损害MoveMe的NT </td></tr><tr><td> 活动 </td><td>正常</td><td>轻度至中度的活性降低</td><td>固定，除非刺激</td></tr><tr><td> 毛皮质地 </td><td>正常</td><td>轻度至中度的皱裂</td><td>严重的皱裂/差疏导</td></tr><tr><td> 肌理 </td><td>正常</td><td>缩放/尾的爪子</td><td>裸露的皮肤明显的地区</td></tr></tbody></table> 表1。Cooke 等人开发的，该评分系统于1996年1。小鼠每天应得分在左边的每个的标准。每个鼠标是为每个标准给予评分为0-2分的总成绩是所有个人得分的总和。 <img alt="图1" src="/files/ftp_upload/3697/3697fig1.jpg" /> 图1。致死剂量照射BALB.B移 ​​植10 7骨单独或与18×10 6 CD8 T细胞的耗尽WT脾细胞和2×10 6个纯化L2G85.B6 CD8 T细胞的骨髓细胞。 A）临床评分数据受助人的骨髓，单独或与CD8 + T细胞耗尽WT脾细胞和纯化L2G85.B6 CD8 + T细胞。 B）生存资料的收件人单独或与骨髓枯竭WT脾细胞和纯化L2G85.B6 CD8 + T细胞CD8 + T细胞。 <a href="http://www.jove.com/files/ftp_upload/3697/3697fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3697/3697fig2.jpg" /> 图2。致死剂量照射BALB.B小鼠的移植与10 7个骨髓细胞单独或与18×10 6 CD8 T细胞的耗尽WT脾细胞和2×10 6个纯化L2G85.B6 CD8 T细胞。收件人分别注射4毫克D-荧光素，通过腹膜内注射，并进行5分钟，在小像素合并使用精诺真IVIS成像。诗紫色表示低强度和高强度区周围绘制整个鼠标和总的通量（光子/秒）进行定量红色代表eudo彩色图像显示。 <img alt="图3" src="/files/ftp_upload/3697/3697fig3.jpg" /> 图3。致死剂量照射BALB.B小鼠的移植与10 7个骨髓细胞单独或与18×10 6 CD8 T细胞的耗尽WT脾细胞和2×10 6个纯化L2G85.B6 CD8 T细胞。收件人分别注射4毫克D-荧光素，通过腹膜内注射，并进行5分钟，在小像素合并使用精诺真IVIS成像。紫色表示低强度，红色代表高强度，伪彩色图像显示。收件人成像A）第4天，B）第6天，C）后第8天转移。

<ol>
	<li>Cooke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+of+idiopathic+pneumonia+syndrome+after+bone+marrow+transplantation:+The+roles+of+minor+H+antigens+and+endotoxin.">An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Berger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+subsets+involved+in+lethal+graft-versus-host+disease+directed+to+immunodominant+minor+histocompatibility+antigens.">T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens.</a> Transplantation. 57, 1095-1102 (1994).</li><li>Nimer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+depletion+of+CD8++cells+for+prevention+of+graft-versus-host+disease+after+bone+marrowtransplantation.+A+randomized+controlled+trial.">Selective depletion of CD8+ cells for prevention of graft-versus-host disease after bone marrowtransplantation. A randomized controlled trial.</a> Transplantation. 57, 82-87 (1994).</li><li>Korngold, R., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+markers+of+T+cells+causing+lethal+graft-vs-host+disease+to+class+I+vs+class+II+H-2+differences.">Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences.</a> Journal of Immunology. 135, 3004-3010 (1985).</li><li>Cao, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+using+labeled+donor+tissues+reveals+patterns+of+engraftment,+rejection,+and+survival+in+transplantation.">Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation.</a> Transplantation. 80, 134-139 (2005).</li><li>Asady, R. E. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-{beta}-dependent+CD103+expression+by+CD8(+)+T+cells+promotes+selective+destruction+of+the+host+intestinal+epithelium+during+graft-versus-host+disease.">TGF-{beta}-dependent CD103 expression by CD8(+) T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease.</a> J. Exp. Med. 201, 1647-1657 (2005).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol Rev. 114, 181-217 (1990).</li><li>Karecla, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+E-cadherin+on+epithelial+cells+by+the+mucosal+T+cell+integrin+alpha+M290+beta+7+(alpha+E+beta+7).">Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin alpha M290 beta 7 (alpha E beta 7).</a> Eur. J. Immunol. 25, 852-856 (1995).</li><li>Cepek, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+between+epithelial+cells+and+T+lymphocytes+mediated+by+E-cadherin+and+the+alpha+E+beta+7+integrin.">Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.</a> Nature. 372, 190-193 (1994).</li><li>Malarkannan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+functional+characterization+of+a+dominant+minor+H+antigen,+H60.">The molecular and functional characterization of a dominant minor H antigen, H60.</a> J. Immunol. 161, 3501-3509 (1998).</li></ol>

没有利益冲突的声明。

这里介绍在小鼠体内诱导移植物抗宿主病的协议小鼠移植物抗宿主病的临床相关的模型。始建于1994年，Berger 等人。的C57BL / 6到BALB.B应变组合是MHC-匹配，移植物抗宿主病（GVHD）的死亡率介导的CD4依赖，CD8 + T效应2，高度相似，临床上最常见的情况3。它是已知的CD8 T细胞移植本身不导致在这个模型中移植物抗宿主病，但是，疾病的进展显着更糟时，CD4 +和CD8 + T细胞相比，CD4 + ...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen公司</td><td> 12633-012 </td><td></td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 10439016 </td><td></td></tr><tr><td> 40μM的细胞过滤</td><td> BD Biosciences公司</td><td> 352340 </td><td></td></tr><tr><td> CD3E-生物素</td><td>德国美天旎生物技术</td><td> 130-093-021 </td><td></td></tr><tr><td>抗生物素微球</td><td>德国美天旎生物技术</td><td> 130-091-147 </td><td></td></tr><tr><td> CD8A微珠</td><td>德国美天旎生物技术</td><td> 130-049-401 </td><td>用于消耗从脾CD8 + T细胞。 </td></tr><tr><td> CD8A纯化抗体鸡尾酒</td><td>德国美天旎生物技术</td><td> 130-095-236 </td><td>用于净化从脾CD8 + T细胞。 </td></tr><tr><td> D-荧光素</td><td>卡尺生命科学</td><td> 122796 </td><td></td></tr></tbody></table>

induction of graft-versus-host disease and in vivo t cell monitoring using an mhc-matched murine model

移植物抗宿主病（GVHD）的广泛使用的骨髓移植作为根治性治疗各种血液不足的限制阻挡。引起的成熟的同种异体反应性T细胞存在于注入收件人的骨髓移植物和移植物抗宿主病到主机器官造成损害。然而，在小鼠中，T细胞，必须添加到骨髓接种物以引起移植物抗宿主病。虽然大量的工作已经做了移植后的T细胞反应的特征，生物发光成像技术是一种非侵入性的方法来监测体内的 T细胞贩运模式。 致命的辐射，受体小鼠移植供体小鼠的骨髓细胞和脾细胞。从L2G85.B6（表达荧光素酶的转基因小鼠）T细胞亚群的移植。只移植特定的T细胞亚群，一个是能够跟踪特定的T细胞亚群在体内 ，并根据他们的位置，发展特异性T​​细胞亚群的作用，在促进移植物抗宿主病在不同时间点的假设。在预定的时间间隔移植后，受体小鼠的是使用精诺真IVIS CCD相机成像的。利用活体成像软件，可量化的光子强度（红色=高强度，紫=低强度）的基础上产生的伪彩色图像的光照强度。 受体小鼠移植后4-7天之间，开始出现临床症状，移植物抗宿主病。 Cooke 等人开发了一个评分系统进行定量疾病进展的基础上的受体小鼠皮毛质地，皮肤的完整性，活动，减肥，和姿势。小鼠每天的得分，和安乐死时，他们变得​​奄奄一息。受鼠一般垂死移植后的20-30天。 小鼠模型，移植物抗宿主病的免疫学研究的有价值的工具。选择性地移植特定的T细胞亚群人仔细辨认每个子集所扮演的角色的新低。非侵入性跟踪在体内的 T细胞反应的价值又增加了一层小鼠移植物抗宿主病（GVHD）模型。

Watch this Scientific Journal Video about Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model at JoVE.com

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

小鼠骨髓移植是一种被广泛使用的技术，研究在人类移植物抗宿主病的免疫机制。监测能力的T细胞贩运模式<em在体内</em&gt;允许在移植物抗宿主病的发展和延续的T细胞反应进行详细的分析。

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 ...

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

15.6K 次观看.  The Ohio State University Medical Center. 小鼠骨髓移植是一种被广泛使用的技术，研究在人类移植物抗宿主病的免疫机制。监测能力的T细胞贩运模式

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

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.

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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 ...

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 ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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 ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...