Name: visualization of il-22-expressing lymphocytes using reporter mice
Uploaded: 2017-01-25T12:30:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

We thank Kelli Czarra and Megan Karwan for animal technical assistance, Kathleen Noer and Roberta Matthai for flow cytometry assistance, and Donna Butcher andMiriam R. Anver for pathology analysis. This project was supported by a grant from the Ely and Edythe Broad Foundation (to Scott Durum) and has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (MRA).

所有动物按照2011指南护理和Frederick国家实验室的实验动物委员会癌症研究使用中所述的实验程序获得适当的照顾。 1.通过代BAC重组工程IL-22-tdTomato记者小鼠注:老鼠应该是无意识的，不响应对有害刺激的运动。消毒用70％乙醇手术区和消毒用玻璃珠灭菌所有外科手术工具。 <ol><li>使用碱性提取方法10，11提纯BAC DNA（RP23-401E11，克隆长度228391个基点）。通过RP23-401E11 5微克的SpeI消化表征BAC克隆以确认靶基因的正确的尺寸。注:BAC DNA被消化成14碎片。 </li><li>将TdTomato报告基因插入PLD53SC-A-GFP-B穿梭载体的不是I /萨尔我的网站11和替换在同一个站点的绿色荧光蛋白基因。然后，使用BAC RP23-401E11为模板，扩增同源框（A和B）到第一翻译外显子IL-22（外显子1）通过PCR（95℃2分钟; 30 95℃30个循环秒，55℃30秒，和72℃30秒;和在72℃最终延伸10分钟）。在一个50μl的PCR反应体系中，添加50纳克的BAC DNA，0.5μl的引物（10μM），40微升的PCR SuperMix中高保真和8微升的H 2 O（1微升） 注:A和B框的引物如下:向前迈进了一箱:5&#39;-牛逼GGCGCGCC GGAGCTGTGAAGAAAG和反向:5&#39;-CAGAGATCGCACAAGTGTCAAC; B寄存器正向:5&#39;-摹TTAATTAA CTGCCCGTCAACA和反向:5&#39;-牛逼GGCCGGCC TGAGCACCTGCTT CATC 10。的限制性内切酶（ASC我和Pac I）的位点有下划线。 </li><li>结扎升序我消化A的500纳克和500纳克的Pac I-消化的B- fragm的进入经济需求测试在16°C 50纳克PLD53SC-ATB（tdTomato）载体的1小时。 </li><li>变换纯化PLD53SC-ATB载体导入BAC-感受态细胞11和培养他们在Luria-BERTANI（LB）琼脂氯霉素（20微克/ ml）和氨苄青霉素（30微克/ ml）的平板，在37℃。从通道/安培主平板挑两至三菌落共整合。 </li><li>接种各菌落在1ml的LB补充有20微克/ ml氯霉素和孵化它们在37℃下1小时，225转。铺在LB-琼脂板100微升每种混合物的（10克蛋白胨140，5克酵母提取物12克琼脂，和1升水的; pH7.5）中补充有氯霉素和蔗糖的4-4.5％。过夜孵育培养在37℃。 </li><li>挑选既大又小的殖民地，replate他们与氯霉素2 LB琼脂平皿上。然后，暴露的板具有或不具有UV光30秒，并挑选用于进一步分析该UV敏感菌落。 </li><li>确定使用tdTomato / IL-22的引物杂用PCR修饰BAC DNA（正向:5&#39;ACT TGT GCG ATC TCT GAT GGT;反向:5&#39;TGT AAT CGG GGA TGT CGG C）。热循环条件如下:95℃2分钟; 95℃30个循环，30秒，56℃30秒，72℃30秒;并在72℃最后延伸10分钟。 </li><li>确认由大制备BAC DNA 12的直接BAC测序的BAC的改性区域的顺序。 </li><li>植入微量注射的受精卵进假孕的C57BL / 6c的小鼠当麻醉接受者小鼠的剂量0.25％三溴乙醇的进入腹膜腔。 </li><li>线性化的IL-22-tdTomato BAC构建（10微克）消化用5微升限制性endonucleasePi-SceI位的在100μl的反应体系和microinject线性的BAC DNA到C57BL / 6雌性受精卵在原核期13。 SCRE烯通过使用标准方法从尾部活检分离的DNA的Southern印迹分析的转基因小鼠。 </li></ol>从脾脏，胸腺，淋巴结和淋巴集结2.单细胞制备方法注:IL-22-tdTomato记者小鼠保持在美国国家癌症研究所（NCI，冯检基，MD）。安乐死方法符合安乐死最新AVMA指引。所有小鼠用CO 2吸入14安乐死。 <ol><li>在CO 2室安乐死的IL-22-tdTomato鼠标并把它放在一块干净的纱布。喷洒70％酒精消毒腹部皮肤，剖开。删除左翼健脾和胸骨后面的胸腺。 </li><li>为了收获mesentericlymph节点（位于肠系膜组织）和淋巴集结（整个小肠的回肠区小lymphoidnodules），取珍珠白肠系膜淋巴结接近到t他用墙解剖先用细点锯齿提示钳，然后取下整个小肠和结肠排出体外的小肠。查找沿肠道扩展椭圆形的补丁（淋巴集结），并用剪刀剪他们。磨这些淋巴组织（淋巴结和淋巴集结）二磨砂载玻片之间单独成在PBS / 1％胎牛血清（FBS）的在冰上缓冲的单细胞悬浮液。 </li><li>剁碎使用相同的方法的脾或胸腺组织如在步骤2.2。离心脾悬浮液在500 xg离心8分钟，4℃。 1毫升每脾的ACK裂解缓冲液加入到细胞沉淀。 </li><li>拌匀，让他们站1分钟。 9毫升无菌磷酸盐缓冲盐水（PBS）缓冲液添加到各样品中。 8分钟降速离心500 XG和4℃以除去红血细胞。 注意:不要添加ACK裂解缓冲胸腺细胞。 </li><li>重悬所得的白细胞在5ml PBS中和p的通过100微米的细胞过滤屁股细胞。收集通过离心细胞沉淀在500 xg离心8分钟。 </li><li>悬浮细胞在5ml RPMI培养基或FACS缓冲液中，并计算用0.4％台盼蓝染料/ PBS溶液中的活细胞。 </li></ol> 3.内淋巴细胞和固有层细胞的分离从肠道<ol><li>打开腹部皮肤如在步骤2.1，切小肠，从十二指肠（旁胃）至回肠（接近附录），和整个结肠，右肛门以上。删除多余的脂肪及肠系膜用组织钳。立即把肠子在冰冷的PBS。 </li><li>寻找淋巴集结（小，粗结节）沿着小肠的远侧区域。取出使用镊子andopen肠道纵向用剪刀群众。彻底冲洗，用10ml冰冷的PBS的肠。 </li><li>孵育组织在20ml预消化缓冲液（5mM EDTA的的和1mM二硫苏糖醇中Hank氏平衡盐溶液（HBSS）中）处理30分钟，在37℃下在摇床缓慢旋转（50rpm下）。每2孵育后，除去上皮细胞层，含有上皮内淋巴细胞（IELs），通过激烈涡旋（在12,000rpm 30秒），并通过一个100微米的细胞滤网通过所有组织中。 </li><li>加入20ml新EDTA溶液添加到组织为第二次温育30分钟，在37℃。通过100微米的细胞滤网通过IEL馏分并与前级分汇集它们。保持其余肠片段在冰上在步骤3.9进行处理。 </li><li>离心汇集IEL分数在800 XG在4℃下5分钟。用10ml的HBSS / 5％FBS的洗涤合并IEL馏分和自旋下来在800 xg离心5分钟，4℃。 </li><li>悬浮细胞在8毫升44％的胶体二氧化硅介质15的并用5ml 67％的胶体二氧化硅介质包裹。离心44％/ 675.5％的细胞混合物梯度，在室温下20分钟，1500×g下。 </li><li>收集从带IEL细胞在界面。首先，删除使用1-毫升吸管将上清液（大约5毫升）的顶层。然后，在胶态二氧化硅介质梯度相间收获IELs。 </li><li>通过加入10ml RPMI培养基洗IELs。旋细胞向下在800 xg离心5分钟，4℃。重悬IELs在5ml RPMI对步骤3.15。 </li><li>对于固有层淋巴细胞的隔离（LPLs），剁碎从步骤3.4肠组织碎片成使用剪刀（1毫米）小片和消化用10ml消化缓冲件（0.05克胶原酶，0.05g的DNA酶I的，并在37℃0.3克在RPMI / 5％FBS的分散酶II）的15-20分钟的。 </li><li>通过70微米的细胞滤网过滤该混合物。流过的上清液中含有的释放LPL。 </li><li>通过重复步骤3.9-3.10（通常完全消化肠片段，它们必须是重复3次）。每一次，集中包含LPLs上清。 </li><li>收集通过离心将细胞在1500 xg离心和室温下5分钟和在RPMI / 5％FBS的悬浮颗粒。传递它们通过40微米的细胞滤网。 </li><li>重悬细胞沉淀在10ml一个40:80胶体二氧化硅介质梯度的40％，级分和在一个15毫升管包裹5毫升80％的级分。 </li><li>在1500 xg离心和室温15执行通过离心密度梯度分离20分钟。 </li><li>收集LPLs，如在步骤3.7中描述。用10毫升的PBS洗一次并在1ml的PBS / 1％FBS的缓冲剂或RPMI培养基悬浮颗粒。 </li><li>算用0.4％台盼蓝染料/ PBS溶液中的活细胞（既IELs和LPLs）。 </li></ol> 4. IL-22 tdTomato在结肠炎的表达<ol><li>以1×10制备浓度从IL-22 TdTomato小鼠单个脾细胞悬浮液&lt;SUP&gt; 7细胞在无菌PBS / ml的，如在步骤2.1-2.4中描述。 </li><li>净化用小鼠的CD4细胞阴性分离方法10的CD4 + T细胞。用抗CD4-APC标记它们（克隆RM4-5，0.5微升/ 1×10 6个细胞在PBS / 1％FBS的缓冲液），抗CD45RB-FITC（克隆16A，0.5微升/ 1×10 6个细胞在PBS中/ 1％FBS的缓冲液）温育在4℃进行30分钟。 </li><li>旋细胞下来，在500 xg离心5分钟，4℃。清洗细胞用2ml PBS / 1％FBS的缓冲液中，在1ml的PBS / 1％FBS的染色缓冲液重悬。 </li><li>运行在流式细胞仪机16流标记的T细胞。门上的T细胞群体的细胞和排序的 CD4 + CD45RB 高双阳性细胞（在488nm和633nm的激光检测的波长）。 </li><li>收获分选的细胞离心，在500 xg离心5分钟，4℃。重悬在无菌PBS缓冲液中的细胞沉淀以1×10 6个 </SUP&gt;细胞/ ml。注入细胞（0.5×10 6个 ）插入腹膜ofeach RAG1 - / -受体小鼠。 </li><li>在不同时间点牺牲CO 2条件下的受体小鼠。收获了肠系膜淋巴结和小肠和大肠，如步骤2.1和2.2描述。把每肠系膜淋巴结和切开肠（纸/肠/纸三明治）在4％低聚甲醛（PFA;在通风橱下处理）过夜以固定的组织。 18％的蔗糖替代PFA 16-24小时，然后冷冻组织切片进行。 </li><li>使用低温恒温器机切割所述组织17，18，19。暖组织切片至室温约60分钟，并放置在丙酮，在室温下10分钟。干他们在室温下大约5分钟。 </li><li> FBS添加过量，用移液管将其删除，并在1添加抗RFP:200稀释在0.1％BSA / 1X PBS。孵育在37℃的抗体60分钟。 </li><li>冲洗在1×PBS中的部分10分钟，相应的二抗（山羊抗兔568）适用于所述组织，稀释1:300在1％BSA / 1×PBS中，在室温下30分钟。 </li><li>冲洗在1X PBS的幻灯片10分钟，擦干晾干，加盖玻片。 </li><li>可视化所有样本下一致的照明荧光显微镜:使用40倍物镜（RFP激发滤光片540-580纳米）。 </li></ol>

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A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+tissues.">Cryosectioning tissues.</a> CSH Protoc. 2008, pdb.prot4991 (2008).</li><li>Hilbe, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+automated+cellular+imaging+system+and+manual+microscopy+for+immunohistochemically+stained+cryostat+sections+of+lung+cancer+specimens+applying+p53,+ki-67+and+p120.">Comparison of automated cellular imaging system and manual microscopy for immunohistochemically stained cryostat sections of lung cancer specimens applying p53, ki-67 and p120.</a> Oncol Rep. 10, 15-20 (2003).</li><li>Suzuki, Y., Furukawa, M., Abe, J., Kashiwagi, M., Hirose, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+porcine+trappin-2+(SKALP/elafin)+in+trachea+and+large+intestine+by+in+situ+hybridization+and+immunohistochemistry.">Localization of porcine trappin-2 (SKALP/elafin) in trachea and large intestine by in situ hybridization and immunohistochemistry.</a> Histochem Cell Biol. 114, 15-20 (2000).</li><li>Wolk, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22+increases+the+innate+immunity+of+tissues.">IL-22 increases the innate immunity of tissues.</a> Immunity. 21, 241-254 (2004).</li><li>Brand, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22+is+increased+in+active+Crohn's+disease+and+promotes+proinflammatory+gene+expression+and+intestinal+epithelial+cell+migration.">IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.</a> Am J Physiol Gastrointest Liver Physiol. 290, G827-G838 (2006).</li><li>Nagalakshmi, M. L., Rascle, A., Zurawski, S., Menon, S., Waal Malefyt, d. e., R, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-22+activates+STAT3+and+induces+IL-10+by+colon+epithelial+cells.">Interleukin-22 activates STAT3 and induces IL-10 by colon epithelial cells.</a> Int Immunopharmacol. 4, 679-691 (2004).</li><li>Maloy, K. J., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+homeostasis+and+its+breakdown+in+inflammatory+bowel+disease.">Intestinal homeostasis and its breakdown in inflammatory bowel disease.</a> Nature. 474, 298-306 (2011).</li><li>Andoh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-22,+a+member+of+the+IL-10+subfamily,+induces+inflammatory+responses+in+colonic+subepithelial+myofibroblasts.">Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts.</a> Gastroenterology. 129, 969-984 (2005).</li><li>Bhan, A. K., Mizoguchi, E., Smith, R. N., Mizoguchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colitis+in+transgenic+and+knockout+animals+as+models+of+human+inflammatory+bowel+disease.">Colitis in transgenic and knockout animals as models of human inflammatory bowel disease.</a> Immunol Rev. 169, 195-207 (1999).</li><li>Mombaerts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+development+of+inflammatory+bowel+disease+in+T+cell+receptor+mutant+mice.">Spontaneous development of inflammatory bowel disease in T cell receptor mutant mice.</a> Cell. 75, 274-282 (1993).</li><li>Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+in+the+intestine:+a+balancing+act+between+effector+and+regulatory+T+cell+responses.">Immune regulation in the intestine: a balancing act between effector and regulatory T cell responses.</a> Ann N Y Acad Sci. 1029, 132-141 (2004).</li><li>Ahlfors, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22+fate+reporter+reveals+origin+and+control+of+IL-22+production+in+homeostasis+and+infection.">IL-22 fate reporter reveals origin and control of IL-22 production in homeostasis and infection.</a> J Immunol. 193, 4602-4613 (2014).</li></ol>

The authors have nothing to disclose.

IL-22在先天宿主防御和组织重塑中起重要作用。 IL生产-22细胞已经鉴定体外通过细胞内染色。然而，它仍然是难以跟踪的IL-22表达的原位 ，无论是在正常状态还是在炎性病症。这个协议描述来开发的IL-22记者小鼠模型中，这使我们能够本地化记者表达细胞在体内的新方法。报告基因编码TdTomato在扰乱的信号序列的位点插入到IL-22的基因座。在报道这种策略的结果被截留在产生细?...

报道基因的细胞类型特异性表达是识别细胞积极地表达于下稳态和摄动态组织中的靶是有用的。它也允许对这些细胞，这保持存活，研究其其它性质的纯化。报告小鼠已被用于在阐明为特定的细胞因子，转录因子，和调节元件的作用机制。先前策略1，2，3在很大程度上依赖于敲记者成在小鼠染色体中，耗时和昂贵的过程中的目标轨迹。因此，对于报告小鼠的生成一个更简单的方法是可取的。 细胞因子是一大类小，分泌的蛋白质/肽调节通过间信号的免疫反应的。白细胞介素22（IL-22）是许多细胞因子报道活动，包括屏障福nction，组织修复和炎症4。虽然IL-22最初被发现作为T细胞产物5，随后的报告证明在人类6和小鼠7和其他类中固有的淋巴细胞8及其在自然杀伤（NK）细胞中表达。尽管IL-22产生细胞的广泛的观察，可视化的IL-22以前需要离体刺激和透的污渍的抗体。因此，新的IL-22的报告小鼠。将研究IL-22的体内平衡和致病过程的功能的非常有用的工具。 在这里，我们开发了一种简化的转基因报告小鼠模型来观察的IL-22产生细胞在体内和体外 。使用BAC重组工程方法9，我们插入的tdTomato cDNA序列与保利的信号碎片进入IL-22 LOC我们和替换外显子1的其它非翻译区，外显子和调控元件没有扰动，因为我们希望尽可能模仿的IL-22的自然调节。记者插入部位扰乱信号序列，从而在生产细胞内的记者的积累，不同于IL-22本身，这是快速分泌。这种新的方法也可以应用到报告小鼠的产生用于其它分泌蛋白。

<table><tbody><tr><td>RP23-401E11 BAC</td><td>Thermo Fisher Scientific</td><td>RPCI23.C</td><td>Need gene ID: 50929</td></tr><tr><td>NucleoBond BAC 100</td><td>Takara Clontech</td><td>740579</td><td></td></tr><tr><td>PCR SuperMix High Fidelity</td><td>Thermo Fisher Scientific</td><td>10790020</td><td></td></tr><tr><td>PI-SceI</td><td>New England Biolabs</td><td>R0696S</td><td></td></tr><tr><td>SpeI</td><td>New England Biolabs</td><td>R0133S</td><td></td></tr><tr><td>LB Broth</td><td>Thermo Fisher Scientific</td><td>10855-001</td><td>1 L: 10 g SELECT Peptone 140, 5 g SELECT Yeast Extract, 5 g sodium chloride&amp;nbsp;</td></tr><tr><td>Anti-mouse CD3</td><td>eBioscience</td><td>11-0031</td><td></td></tr><tr><td>Anti-mouse CD4</td><td>eBioscience</td><td>17-0041</td><td></td></tr><tr><td>Anti-mouse CD45</td><td>Thermo Fisher Scientific</td><td>MCD4530</td><td></td></tr><tr><td>Anti-mouse CD45RB</td><td>eBioscience</td><td>11-0455</td><td></td></tr><tr><td>Anti-mouse RFP</td><td>Abcam</td><td>Ab62341</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Thermo Fisher Scientific</td><td>14175145</td><td>KCl, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, NaHCO&lt;sub&gt;3&lt;/sub&gt;, NaCl, D-Glucose</td></tr><tr><td>Dnase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Thermo Fisher Scientific</td><td>A1049201</td><td></td></tr><tr><td>Percoll</td><td>GE healthcare life sciences</td><td>17-0891-01</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088858001</td><td></td></tr><tr><td>Dispase II (neutral protease, grade II)</td><td>Roche</td><td>4942078001</td><td></td></tr><tr><td>IX70 inverted fluorescence microscope</td><td>Olympus</td><td></td><td>Ask for quote</td></tr><tr><td>Nikon Eclipse 80i microscope</td><td>Nikon</td><td></td><td>Ask for quote</td></tr><tr><td>Dynal shaker</td><td>Electron Microscopy Science</td><td>61050-10</td><td></td></tr><tr><td>FACSAria</td><td>BD Bioscience</td><td></td><td>Ask for quote</td></tr><tr><td>LSRII SORP/flow cytometry</td><td>Becton, Dickinson and Company&amp;nbsp;</td><td></td><td>Ask for quote</td></tr></tbody></table>

visualization of il-22-expressing lymphocytes using reporter mice

报告小鼠已被广泛用于观察目标基因的表达的定位。该协议的重点是建立一个新的转基因记者小鼠模型的战略。我们选择了可视化白介素（IL）22的基因表达，因为这种细胞因子具有在肠道内，在那里它有助于修理由炎症损伤组织的重要活动。记者系统提供优于识别产品在体内的其它方法相当大的优势。在IL-22的情况下，其他的研究已经首次分离自组织的细胞，然后再刺激在体外细胞中。 IL-22，这通常是分泌的，使用一种药物被困在细胞内，并用于细胞内染色来可视化它。本方法识别能够产生的IL-22的细胞，但并不确定它们是否在体内做。记者设计包括以这样的洼插入基因为荧光蛋白（tdTomato）插入IL-22的基因该荧光蛋白不能被分泌，因此ÿ保持截留在体内产生细胞内。荧光生产者可以然后在组织切片或通过流式细胞仪体外分析来可视化。对于记者的实际施工过程中包括重组工程包含了IL-22基因的细菌人工染色体。然后，该工程化的染色体被引入到小鼠基因组。在不同的小鼠组织中，包括脾，胸腺，淋巴结，淋巴集结，以及肠，通过流式细胞术分析中观察到稳态的IL-22报告表达。结肠炎诱导T细胞（CD4 + CD45RBhigh）转让，并可视化报告基因的表达。阳性T细胞首先出现在肠系膜淋巴结，然后将它们累加远端小肠和结肠组织的固有层中。相比于IL-22 EXPRES使用BAC的策略得到了良好的高保真记者表达锡永，并且它比敲入程序简单。

鼠的IL-22报告基因使用重组工程修改细菌人工染色体携带IL-22的轨迹创建。 图1示出含有sacBII基因，正选择标记物，和氯霉素抗生素抗性基因11 pBACe3.6矢量的图。引入tdTomato到外显子1后，信号肽序列被打乱， 如图2。因此，tdTomato记者被困的IL-22表达细胞内，使它们的检测和隔离通过流式细胞术。纯合子小鼠购自创始人线育成?...

Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

visualization-of-il-22-expressing-lymphocytes-using-reporter-mice

Research

JoVE Journal

Immunology and Infection

8.8K 次观看.  National Cancer Institute, National Institutes of Health. We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse. 

视频: Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

可行的小鼠IL - 17分泌性T细胞的检测和隔离

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

科研

生物学

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

免疫与感染

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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 ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

李斯特菌单细胞基因感染后小鼠脾脏原位干扰素伽马生产的成像

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...