This work was supported by EU Marie Curie Fellowship (EU/300686-InfO) to S.B. and a Research Prize from the United European Gastroenterology Foundation to H.C. We would like to thank Harry Begthel, Jeroen Korving and the Hubrecht Imaging Center for technical assistance, Meritxell Huch for help with initial organoid culture and Yana Zavros for discussion.

1.建立胃化培养的注意:此协议可用于从鼠或人组织胃腺的隔离。它建议使用约1平方厘米的组织。人体组织可以从胃切除术或活组织检查获得。 <ol><li>材料的制备 注意:使用地下室矩阵是基底膜。置于冰上地下室矩阵在任何时候。存储地下室基质在-20℃并在使用前解冻冰上。基础培养基是指高级的DMEM / F12补充有HEPES;适当的谷氨酰胺源如Glutamax的和适当的抗生素，如1×Primocin。所用的培养箱是一个标准的细胞培养孵化器中（37℃，湿润气氛，5％的CO 2） <ol><li>锋利的钳子，剪刀，手术刀和玻璃显微镜载玻片:通过高压灭菌或用100％异丙醇消毒准备餐具。 </li><li>隔离的前一天，放置24 WEL升细胞培养板的孵化器。 </li><li>根据制造商的建议，准备地下室矩阵。准备1毫升等分，以避免反复冻融循环。 </li><li>制成500毫升之螯合缓冲和冷静的冰。制备1×缓冲液从灭菌蒸馏水以5.6毫的 Na 2 HPO 4，8.0毫KH 2 PO 4，96.2毫摩尔NaCl，1.6毫米氯化钾，43.4毫蔗糖，54.9毫D-山梨醇，0.5mM的DL-二硫苏糖醇（注意），pH值7.如果计划数隔离，没有做好准备的DL-二硫5倍浓缩液。无菌过滤器的缓冲器。隔离之前，稀释5倍的缓冲用无菌水，以1x和刚分离之前添加DL-二硫苏糖醇。 </li><li>准备并根据制造商的建议冲淡一切的生长因子。使用小尺寸的等分试样，并避免反复冻融。官能生长因子是对结果的关键。 </li><li>在冰上凉爽基础培养基并冷却离心机到46℃。 </li><li>暖基础培养基的另一等分试样至37℃。 </li></ol></li><li>腺的分离<ol><li>在冰冷的培养基收集大约1平方厘米的组织。置于冰上，直到准备。虽然这是优选尽快处理组织，在介质商店组织在冰上如果必要的。 </li><li>将组织在一个10cm培养皿并用冷的1×螯合缓冲器覆盖。轻轻来回移动清洗。 </li><li>将组织在干燥10cm培养皿。使用镊子，小心地取出在立体显微镜下粘液以及肌肉层。轻轻来回移动用冷1×螯合缓冲器的组织。 </li><li>地点1 平方厘米的组织在一个新的，干燥的10cm培养皿。用剪刀把它切成20-50小块​​约2-5平方毫米大小。 </li><li>使用镊子，将所有片放入5​​0毫升管中。 </li><li>取10ml无菌塑料吸管。潮湿1X螯合缓冲吸液管。保持使用整个过程中同样的吸管。 </li><li>加入10毫升冷的1×螯合缓冲器向50ml管中。通过大力吹打向上和向下10倍洗片。让片安定。去除上清。 </li><li>重复洗涤（步骤1.2.7），直到上清液是明确的。这1平方厘米的人体组织大约需要1个螯合缓冲50-100毫升。 </li><li>孵育组织片在20ml 1×螯合缓冲补充有10毫摩尔EDTA于室温进行10分钟，轻轻颠倒每2分钟。 注意:时间孵育的温度和可为任何一个的速度和/或保存腺体结构的最优化。它们的范围可以从几个小时的孵育在4℃下在滚动平台至5分钟，在37℃，在用200rpm的摇床。很好的出发点为小鼠胃是30分钟，在4℃下在滚动平台和人胃，在室温10分钟。 </li><li>吸取一次轻轻地上下用10毫升吸管。允许PI欧洲经委会的解决。弃去上清液。 </li><li>使用移液管，将片转移到一个干净10cm培养皿。除去液体。 </li><li>放置在组织片的顶部上的玻璃显微镜载玻片上。观察倒置显微镜与放大10倍的细胞培养下的完整的组织。 </li><li>施加压力，在载玻片和培养皿。同时按住手（带无菌手套），并按下玻璃用拇指。所述组织的周缘将出现混浊表明腺体释放出来的组织。 </li><li>观察倒置显微镜与放大10倍的细胞培养下发布的腺体。 </li><li>收集腺体及组织在30毫升冷的基础培养基。让大型组织碎片定居。 </li><li>收集含腺体上清两个15ml试管。 </li><li>离心5分钟，在200×g离心并4℃。弃上清。该颗粒包含分离的腺体。让他们在冰上。 </li></ol></li><li>腺的播种<ol&gt; <li>对于播种，保持地下室矩阵冰冷的时刻，所以它会留液体。 </li><li>方案1:种子6口井的稀释排。 <ol><li>暂停在60微升地下室矩阵的沉淀。 </li><li>准备5无菌1.5毫升管在冰上用每个含有50微升的地下室矩阵。转移10微升腺体/地下室基质悬浮液进入第一1.5毫升管。暂停腺体和转移10微升的该溶液到下一个1.5毫升的试管。重复下一个管。 </li></ol></li><li>选择2:每孔种子腺体的计算量。开始在24孔板的一个孔每50μl地下室矩阵100腺体。 <ol><li>仔细暂停在10ml基本培养基中的沉淀腺体。将50微升放在一个干净的10毫升培养皿。数倒置显微镜与放大10倍的细胞培养下腺体的数量。 </li><li>计算在溶液腺体的浓度。转移体积CONTA插件400腺体到一个新的15ml试管。 </li><li>离心5分钟，在200×g离心并4℃。弃上清，保持管在冰上。加入200μl地下室矩阵，仔细悬浮。置于冰上。 </li></ol></li><li>采取37℃的温水24孔板（见步骤1.1.2）从培养箱。放置一滴50μl的腺体地下室矩阵在阱的中央。液滴就会形成圆顶。 </li><li>板小心地转移回细胞培养孵化器。让地下室矩阵固化10分钟。 </li><li>准备御寒中所有的生长因子。 <ol><li>人类腺:有以下的生长因子对人胃癌组织体补充基础培养基:50毫微克/毫升的EGF，10％的noggin条件培养基，10％的R-spondin1条件培养基，50％的Wnt-条件培养基，200纳克/ ml成纤维细胞生长因子（FGF）10，1nM的胃泌素，和2μMTGFbeta抑制剂和10μMRho相关卷曲螺旋形成蛋白丝氨酸/苏氨酸激酶<html>抑制剂（RHOKi）。 </li><li>对鼠腺:有以下的生长因子对小鼠胃组织体补充基础培养基:50毫微克/毫升的EGF，10％的noggin条件培养基，10％的R-spondin1条件培养基，50％的Wnt-条件培养基，100纳克/毫升FGF10，10nM的胃泌素，0.5毫TGFbeta抑制剂和10μMRHOKi。 </li></ol></li><li>就拿板的孵化器。小心加入所有的生长因子（1.3.6），以每500微升介质良好，无干扰的地下室矩阵。将其放回孵化器。 </li><li>刷新每周介质3次（每2-3天）。对于刷新的媒体，它留下的地下室矩阵的下降完好删除旧的介质。小心地加入新鲜，温暖中所有的生长因子。不要添加RHOKi。只有后腺体播种直接或类器官（步骤2）的分割后添加RHOKi。 </li></ol></li></ol> 2.胃通道化培养的显微注射前。 ove_content"&gt;注:每种类型化培养都有自己的倍增时间小鼠肠道和胃的文化通常被分成1:5每5-7天人类肠道文化被分割1:5每隔10-12天人类胃的文化被分成1:5每14天，如果从单个细胞开始，人类的胃组织体也可能需要20天的正常形成，是一个好兆头，如果崭露头角结构环绕中心腔在这个协议中，类器官被分成。 4孔板用于显微注射。维护组织体遵循相同的协议，但可以使用任何其他的细胞培养板，如标准的24孔细胞培养板。 <ol><li>材料的制备<ol><li>高压灭菌巴氏吸管。 </li><li>一天通道之前，放置4孔细胞培养板在孵化器在37℃。 4孔板具有低边和允许显微操作。 </li><li>根据制造商的建议，准备地下室矩阵。上passagi的天NG，解冻地下室矩阵冰上。 </li><li>准备并根据制造商的建议冲淡一切的生长因子。 </li><li>在冰上凉爽基础培养基并冷却离心机至4℃。 </li></ol></li><li>类器官文化通道<ol><li>就拿板从孵化器中的组织体。从一个孔中取出介质。 </li><li>加入1毫升冷基础培养基，以在阱地下室矩阵。使用1ml微量，大力吸管上下直至地下室矩阵打破。转移到15毫升管。放置在冰上。 </li><li>拿一个玻璃巴斯德吸管，并为本生​​灯火源等。 <ol><li>握住吸管尖在火直到针尖的开口已经从1.5毫米缩小到约0.5毫米（ 图1）。让吸管冷却至室温。 </li><li>潮湿基础培养基的吸管。一个最佳收窄吸管会占用介质明显比未缩小吸管慢，但它还是会所有流移液好。 </li></ol></li><li>拿起类器官中的吸管，大力吸液管10倍上下。的组织体的断裂可通过眼睛观察到。 </li><li>添加9ml冷基础培养基和离心5分钟，在200×g离心并4℃。 </li><li>小心丢弃所有上清。暂停在250微升地下室基质沉淀。置于冰上。 </li><li>以37℃的温水4孔培养板，并放置在每个下降了50微升类器官/地下室矩阵很好。 </li><li>转移板仔细回孵化器。让地下室基质固化，在37℃下进行10分钟。 </li><li>备的小鼠或人类器官适当介质如在步骤1.3.6列出。 </li><li>就拿板的孵化器，并覆盖500微升中每个地下室矩阵下降。 </li><li>板转移到孵化器。 </li><li>刷新介质，每周3次，每2-3天（见1.3.8。）。 </li><li>对于组织体冻结，mechanically破坏类器官如本协议描述的分割（步骤2.2.1-2.2.5）。暂停在500毫升冷冻介质中的组织体碎片和冷冻至-80℃，用冷冻管和冷冻集装箱。对于长期储存，将管转移到液氮中。 </li><li>对于组织体解冻，如步骤1.1.2，1.1.3，1.1.5，1.1.6，1.1.7概述准备培养板，地下室基质，生长因子，冷暖网上平台。解冻到15ml管，用10毫升冷基础培养基后立即暖冷冻瓶使用水浴，和转移的细胞。离心机和板细胞在2.2.5概述为2.2.12。 注:组织体的显微注射是费力，但允许独特的机会来研究互动3D。 </li><li>如果不需要三维研究，种子组织体在两个维度。为此，扰乱类器官如步骤2.2.1到2.2.6布局。所有生长因子加入2250微升冷介质250微升地下室马TRIX与类器官片段。 <ol><li>种子在5个孔24孔板中，每孔500微升。改变介质每周3次，用小心地仅更换上部400微升，以保持基底基质在板不受干扰的底部。 注意:类器官会附着在细胞培养板和扩大在2D。电子显微镜表明，细胞的顶侧面中的孔的介质（数据未显示）。 </li><li>感染前，所有交换介质，包括地下室矩阵，以允许细菌附着到细胞中。类似的协议也被感染描述H.幽门螺杆菌 14。二维的细胞层可以含有不存在于三维组织体的所有细胞类型。 </li></ol></li></ol></li></ol>化培养的3显微注射。 注意:此协议可用于microinject细菌进入组织体。它可能有助于启动注射类器官这是更宽容，以显微注射。例如，鼠标胃组织体可以长到非常大的囊性类器官，很容易的目标。 <ol><li>材料的制备<ol><li>拉玻璃毛细管成使用微量牵拉两个注射针，根据制造商的建议。的微量拉马将加热在中间的玻璃毛细管，将拉毛细管成两部分，从而产生两个玻璃针。它是实用拉几个针，并将它们存储在一个干净的培养皿中。 </li><li>以允许细菌感染，来自所有媒体取出抗生素至少3介质的变化（=一周）。这将稀释从地下室矩阵抗生素。如果使用的细菌菌株是抗特定抗生素，添加该特定的抗生素，以减少造成污染的机会。 </li><li>准备工作台上进行显微注射​​。在不同的生物安全水平工作，可能有必要将一个立体声显微镜无菌工作台内。在这个协议中，注入H.在生物安全级别下的无菌工作台内的立体显微镜2的条件幽门螺旋杆菌 。 </li><li>培养根据标准方法13,23细菌。 </li><li>为了估计感染复数（MOI）的多重性，估计两个参数:细菌的数量和如下所述每类器官的细胞数。教学语言可以与结果相关，例如与用户主机的IL-8的表达13。 <ol><li>来计算细菌密度在感染溶液，首先建立光密度和集落形成单位的根据标准方法24标准曲线（CFU）。 0.1在550nm处的光密度应该产生约10 7 CFU /毫升。 </li><li>来估计每类器官的细胞数，计算在1类器官的数目良好。扰乱类器官如步骤概述2.2.1-2.2.4。后离心5分钟，在200×g离心，4℃，加入1毫升酶解液，重悬。 <ol><li>孵育在37℃，反复振摇5-10分钟。确定是否细胞已（在放大10倍倒置显微镜，如徕卡DMIL）分解成显微镜下的单细胞。如果需要延长孵化。 </li><li>计数使用血球每类器官的细胞，并计算每组织体细胞的数目。人胃组织体的直径为约200μm有大约4,000个细胞。注射0.2微升的细菌溶液每毫升结果1×10 9细菌在50近似MOI。 </li></ol></li></ol></li></ol></li><li>注射组织体。 注意:对于显微注射，玻璃针被稳定在一个保持器，其可以通过显微操作被操纵3维。类器官保持在井的地下室基质内。由于尺寸和定位的好，并不是所有的组织体是同样一个menable注射。通常，在一个良好的30家最大的组织体可以有针对性在5分钟内。 <ol><li>根据标准方案24在基础培养基收获细菌并把它们洗干净。 </li><li>计算每根据光密度毫升细菌的数量（见步骤3.1.5.1）。稀释细菌每毫升1×10 9个细菌在基础培养基。 </li><li>拿一个玻璃针。使用镊子打破尖端，使得开口将约为10微米宽。 </li><li>针插入的注射保持器，并将其固定在微操作器。 </li><li>拿起约10微升菌液进针。 </li><li>将一个4孔培养板，低边包含了体视显微镜下的类器官文化。 </li><li>与显微导航，目标单一类器官用针。定位针靠近类器官，然后将针头插入与一快速运动的组织体。注入approximatel用微量Ÿ0.2微升菌液到每个类器官。 </li></ol></li><li>嘉实类器官的任何分析。 <ol><li>作为例子，4小时后修复类器官。除去上清液，加入1毫升2％的甲醛，并培育20分钟，在室温下或O / N在4℃。组织体可以根据标准程序25进行处理用于免疫组织化学。 </li></ol></li></ol>

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	<li>Aiuto, L., 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=Human+Induced+Pluripotent+Stem+Cell-Derived+Models+to+Investigate+Human+Cytomegalovirus+Infection+in+Neural+Cells.">Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells.</a> PLoS ONE. 7 (11), e49700 (2012).</li><li>Penkert, R. R., Kalejta, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Embryonic+Stem+Cell+Lines+Model+Experimental+Human+Cytomegalovirus+Latency.">Human Embryonic Stem Cell Lines Model Experimental Human Cytomegalovirus Latency.</a> mBio. 4 (3), e00298-13-e00298-13 (2013).</li><li>Roelandt, 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=Human+pluripotent+stem+cell-derived+hepatocytes+support+complete+replication+of+hepatitis+C+virus.">Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus.</a> J Hepatol. 57 (2), 246-251 (2012).</li><li>Schwartz, R. E., Trehan, 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=Modeling+hepatitis+C+virus+infection+using+human+induced+pluripotent+stem+cells.">Modeling hepatitis C virus infection using human induced pluripotent stem cells.</a> Proc Natl Acad Sci USA. 109 (7), 2544-2548 (2012).</li><li>Shlomai, 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=Modeling+host+interactions+with+hepatitis+B+virus+using+primary+and+induced+pluripotent+stem+cell-derived+hepatocellular+systems.">Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems.</a> Proc Natl Acad Sci USA. 111 (33), 12193-12198 (2014).</li><li>Wu, X., 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=Productive+Hepatitis+C+Virus+Infection+of+Stem+Cell-Derived+Hepatocytes+Reveals+a+Critical+Transition+to+Viral+Permissiveness+during+Differentiation.">Productive Hepatitis C Virus Infection of Stem Cell-Derived Hepatocytes Reveals a Critical Transition to Viral Permissiveness during Differentiation.</a> PLoS Pathogens. 8 (4), e1002617 (2012).</li><li>Yoshida, T., 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=Use+of+human+hepatocyte-like+cells+derived+from+induced+pluripotent+stem+cells+as+a+model+for+hepatocytes+in+hepatitis+C+virus+infection.">Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection.</a> Biochem Biophys Res Commun. 416 (1-2), 119-124 (2011).</li><li>Ng, 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=Human+iPSC-Derived+Hepatocyte-like+Cells+Support+Plasmodium+Liver-Stage+Infection+In+Vitro.">Human iPSC-Derived Hepatocyte-like Cells Support Plasmodium Liver-Stage Infection In Vitro.</a> Stem Cell Report. 4 (2), (2015).</li><li>Klotz, C., Aebischer, T., Seeber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+cell+cultures+and+organoids+for+protozoan+parasite+propagation+and+studying+host-parasite+interaction.">Stem cell-derived cell cultures and organoids for protozoan parasite propagation and studying host-parasite interaction.</a> Int J Med Microbiol. 302 (4-5), 203-209 (2012).</li><li>Engevik, M. 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=Loss+of+NHE3+alters+gut+microbiota+composition+and+influences+Bacteroides+thetaiotaomicron+growth.">Loss of NHE3 alters gut microbiota composition and influences Bacteroides thetaiotaomicron growth.</a> AJP: GI. 305 (10), G697-G711 (2013).</li><li>Wilson, S. S., Tocchi, A., Holly, M. K., Parks, W. C., Smith, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+intestinal+organoid+model+of+non-invasive+enteric+pathogen-epithelial+cell+interactions.">A small intestinal organoid model of non-invasive enteric pathogen-epithelial cell interactions.</a> Mucosal Immunol. 8 (2), 352-361 (2015).</li><li>McCracken, K. 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=Modelling+human+development+and+disease+in+pluripotent+stem-cell-derived+gastric+organoids.">Modelling human development and disease in pluripotent stem-cell-derived gastric organoids.</a> Nature. 516 (7531), 400-404 (2014).</li><li>Bartfeld, 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=In+Vitro+Expansion+of+Human+Gastric+Epithelial+Stem+Cells+and+Their+Responses+to+Bacterial+Infection.">In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection.</a> Gastroenterology. 148 (1), (2014).</li><li>Schlaermann, P., Toelle, B., 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=A+novel+human+gastric+primary+cell+culture+system+for+modelling+Helicobacter+pylori+infection+in+vitro.">A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.</a> Gut. , (2014).</li><li>Bertaux-Skeirik, N., 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=CD44+Plays+a+Functional+Role+in+Helicobacter+pylori-induced+Epithelial+Cell+Proliferation.">CD44 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation.</a> PLOS Pathogens. 11 (2), e1004663 (2015).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt+villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Sato, T., 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=Long-term+Expansion+of+Epithelial+Organoids+From+Human+Colon,+Adenoma,+Adenocarcinoma,+and+Barrett's+Epithelium.">Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Barker, N., 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=Lgr5+ve+Stem+Cells+Drive+Self-Renewal+in+the+Stomach+and+Build+Long-Lived+Gastric+Units+In+Vitro.">Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro.</a> Cell Stem Cell. 6 (1), 25-36 (2010).</li><li>Huch, M., 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=In+vitro+expansion+of+single+Lgr5++liver+stem+cells+induced+by+Wnt-driven+regeneration.">In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.</a> Nature. 494 (7436), 247-250 (2013).</li><li>Huch, M., 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=Long-Term+Culture+of+Genome-Stable+Bipotent+Stem+Cells+from+Adult+Human+Liver.">Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver.</a> Cell. 160 (1-2), 299-312 (2015).</li><li>Boj, S. F., 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=Organoid+Models+of+Human+and+Mouse+Ductal+Pancreatic+Cancer.">Organoid Models of Human and Mouse Ductal Pancreatic Cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Karthaus, W. R., 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=Identification+of+multipotent+luminal+progenitor+cells+in+human+prostate+organoid+cultures.">Identification of multipotent luminal progenitor cells in human prostate organoid cultures.</a> Cell. 159 (1), 163-175 (2014).</li><li>Bartfeld, 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=High-throughput+and+single-cell+imaging+of+NF-kappaB+oscillations+using+monoclonal+cell+lines.">High-throughput and single-cell imaging of NF-kappaB oscillations using monoclonal cell lines.</a> BMC cell. 11, 21 (2010).</li><li>Blanchard, T. G., Nedrud, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Maintenance+of+Helicobacter+Species.">Laboratory Maintenance of Helicobacter Species.</a> Curr Protoc Microbiol. , (2006).</li><li>Van Es, J. H., de Geest, N., van de Born, M., Clevers, H., Hassan, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+stem+cells+lacking+the+Math1+tumour+suppressor+are+refractory+to+Notch+inhibitors.">Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors.</a> Nat Commun. 1 (2), 1-5 (2010).</li><li>Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Video+Protocol+of+Retroviral+Infection+in+Primary+Intestinal+Organoid+Culture.">A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture.</a> J Vis Exp. (90), (2014).</li><li>Stange, D. E., Koo, B. -. 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=Differentiated+Troy++Chief+Cells+Act+as+Reserve+Stem+Cells+to+Generate+All+Lineages+of+the+Stomach+Epithelium.">Differentiated Troy+ Chief Cells Act as Reserve Stem Cells to Generate All Lineages of the Stomach Epithelium.</a> Cell. 155 (2), 357-368 (2013).</li><li>Van de Wetering, M., Sancho, E., 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=The+beta-catenin/TCF-4+complex+imposes+a+crypt+progenitor+phenotype+on+colorectal+cancer+cells.">The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.</a> Cell. 111 (2), 241-250 (2002).</li><li>Schumacher, M. A., Aihara, E., 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=The+use+of+murine-derived+fundic+organoids+in+studies+of+gastric+physiology.">The use of murine-derived fundic organoids in studies of gastric physiology.</a> Journal Physiol. 593 (8), 1809-1827 (2015).</li><li>Schwank, G., Andersson-Rolf, A., Koo, B. -. K., Sasaki, N., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+BAC+Transgenic+Epithelial+Organoids.">Generation of BAC Transgenic Epithelial Organoids.</a> PLoS ONE. 8 (10), e76871 (2013).</li><li>Koo, B. -. 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=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nat Meth. 9 (1), 81-83 (2012).</li><li>Schwank, G., 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=Functional+repair+of+CFTR+by+CRISPR/Cas9+in+intestinal+stem+cell+organoids+of+cystic+fibrosis+patients.">Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.</a> Cell Stem Cell. 13 (6), 653-658 (2013).</li><li>Li, V. S. W., Ng, S. 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=Wnt+Signaling+through+Inhibition+of+&#946;-Catenin+Degradation+in+an+Intact+Axin1+Complex.">Wnt Signaling through Inhibition of &#946;-Catenin Degradation in an Intact Axin1 Complex.</a> Cell. 149 (6), 1245-1256 (2012).</li><li>Van de Wetering, M., 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=Prospective+derivation+of+a+'Living+Organoid+Biobank'+of+colorectal+cancer+patients.">Prospective derivation of a 'Living Organoid Biobank' of colorectal cancer patients.</a> Cell. 161 (4), 933-945 (2015).</li></ol>

Hans Clevers is an inventor on several patents for organoid culture. Otherwise the authors have nothing to disclose.

This protocol describes the use of ever-expanding, untransformed primary organoids from adult stem cells for infection biology. Critical steps are i) the isolation of viable glands, ii) expansion of organoids and iii) the microinjection. Below are some suggestions for modifications, troubleshooting and technical considerations. 
Compared to other isolation methods, which use vigorous shaking or pipetting to release glands and can be equally successful, the technique presented here has the adva...

病原体的研究依赖于适当的模型系统来模拟体内的感染。对于一些感染剂，适当的模型系统缺乏而一些使用的系统是远非最佳。一个例子是在胃幽门螺杆菌 （ 幽门螺杆菌 ），这是因果相关的胃癌的发展。然而，在不存在一个更合适的细胞培养体系，许多研究旨在分析癌症发展使用癌细胞系的分子机制，这代表了癌变级联的端点。伯，非转化细胞。将这些研究更好的模型。然而，原代细胞仅可从少数献血者的，并且不能在较长的时间周期中培养。近年来，干细胞研究取得了显著的进步，为原代细胞培养感染生物学研究新的来源。 从文化3干细胞来源已被用于:胚胎干细胞（ESC），诱导多能干细胞（IPSC）或成体干细胞。它们已被用于模拟感染的病毒，如巨细胞病毒1,2-或丙型肝炎病毒3 - 7，寄生虫，如恶性疟原虫 8或 弓形虫 9，和细菌，如Bacterioides thetaiotaomicron 10或肠道沙门氏菌 11。最近，一些方法已经被发布到感染H.模型幽门螺杆菌与来自ESC键或iPS细胞12，小鼠成体干细胞21,22或人类成体干细胞衍生的13类器官- 15。 类器官培养物从成体干细胞的发育源自研究，其中单个干细胞从鼠肠上皮分离接种到3维矩阵和嵌入在模仿含有EGF的有丝分裂原，R-脊椎蛋白的肠干细胞的环境，以提高Wnt信号和头蛋白抑制骨形态发生蛋白（BMP）信令16培养基。值得注意的是这些培养不需要共培养间充质细胞。在这些条件下，干细胞增殖并形成小的结构与结构域窝藏肠隐窝的细胞，以及包含肠绒毛的细胞结构域。的组织体从而自我组织以模拟体内情况 。今天，许多鼠和人性化的组织的成体干细胞可以在体外和生长自组织成类器官类似于其在体内对应物，如小肠和结肠17，胃13,18，19,20肝，胰腺和21前列腺22。 在这里，我们提供了一个视频协议，以培养小鼠和人胃癌组织体的成体干CELLS和microinject它们与H.幽门螺旋杆菌 。该协议是基于以往的报告13,18。该方法可适于用于培养和感染其他类器官培养物如肠组织体。

<table border="0">
	<tbody>
		<tr>
			<td>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, GFR, phenol free</td>
			<td>BD</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-079</td>
			<td>Stock concentration 200 mM, final concentration 2 mM</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>Stock concentration 50 x, final concentration 1x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td>Stock concentration 500 mM, final concentration 1 mM</td>
		</tr>
		<tr>
			<td>Murine recombinant EGF</td>
			<td>Invitrogen</td>
			<td>PMG8043</td>
			<td>Stock concentration 500 &#181;g/mL, final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>Human recombinant FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>Stock concentration 100 &#181;g/mL, final concentration 200 ng/mL</td>
		</tr>
		<tr>
			<td>TGF&#946;i A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>Stock concentration 500 &#181;M, final concentration 2 &#181;M&#160;</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td>Stock concentration 1 M, final concentration 10 mM&#160;</td>
		</tr>
		<tr>
			<td>[Leu15]-Gastrin</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td>Stock concentration 100 &#181;M, final concentration 1 nM</td>
		</tr>
		<tr>
			<td>RHOKi Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503</td>
			<td>Stock concentration 10 mM, final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>Wnt3A conditioned medium</td>
			<td></td>
			<td></td>
			<td>Stable cell line generated in the Clevers Lab. Final concentration 50%. Cells can be obtained from Hans Clevers.</td>
		</tr>
		<tr>
			<td>R-spondin1 conditioned medium</td>
			<td></td>
			<td></td>
			<td>Stable cell line generated in the Kuo Lab. Final concentration 10%. Cell line can be obtained from Calvin Kuo, Stanford.</td>
		</tr>
		<tr>
			<td>Noggin conditioned medium</td>
			<td></td>
			<td></td>
			<td>Stable cell line generated in the Clevers Lab. Final concentration 10%. Cells can be obtained from Hans Clevers.</td>
		</tr>
		<tr>
			<td>R-spondin3</td>
			<td>R&#38;D</td>
			<td>3500-RS/CF</td>
			<td>Alternative source for R-spondin. This has been tested on human intestine organoids (1 &#181;g/mL), but not yet on gastric organoids.</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10</td>
			<td>Alternative source for noggin. This has been tested on human intestine organoids (100 ng/mL), but not yet on gastric organoids.</td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Life Technologies</td>
			<td>12605036</td>
			<td>Enzymatic dissociation solution&#160;</td>
		</tr>
		<tr>
			<td>CoolCell&#174; Alcohol-Free Cell Freezing Containers</td>
			<td>biocision</td>
			<td>BCS-405</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery Cell Culture Freezing Medium</td>
			<td>Invitrogen</td>
			<td>12648-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td>An antibiotics composition agains bacteria and fungi. It is helpful after initiation of a culture. For long term culture you can switch to other antibiotics or none.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Stock concentration 10000/10000 U/mL, final concentration 100/100 U/mL. Can be used alternatively to Primocin in long term culture.</td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Neolabs</td>
			<td>2-1033</td>
			<td>Tweezers with fine tips are helpful for the removal of muscle layer from the tissue.</td>
		</tr>
		<tr>
			<td>4 Well Multidishes</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td>You can use other Multidishes. These were particularly helpful for microinjections because they have a low outer rim and allow more mobility for the manipulator.</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM-5B</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ75</td>
			<td></td>
		</tr>
		<tr>
			<td>Workbench</td>
			<td>Clean Air</td>
			<td></td>
			<td>Custom made to fit the stereomicroscope in ML2 condition</td>
		</tr>
		<tr>
			<td>Cappillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC100T-10</td>
			<td>1 mm outer diameter, 0,78 mm inner diameter.</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>Flaming Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr>
			<td>anti Cag A antibody</td>
			<td>Santa Cruz</td>
			<td>sc-25766</td>
			<td></td>
		</tr>
	</tbody>
</table>

organoids as model for infectious diseases: culture of human and murine stomach organoids and microinjection of helicobacter pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

该协议允许胃腺（图2）的隔离。腺接种入地下室矩阵，它凝固成内井降，提供一个三维框架富含层粘连蛋白和胶原蛋白，以允许腺体生长成组织体（ 图3）。类器官通常开始为小囊肿和内12日至16日，它们扩展成球形的直径为50-300微米（图4）。有些组织体将留囊肿，有的会发展小buddings。后者通常是一个健康的生长培养的标志。在这个协议中一个24孔板的一个?...

Watch this Scientific Journal Video about Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori at JoVE.com

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病:人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

organoids-as-model-for-infectious-diseases-culture-human-murine

Research

JoVE Journal

Immunology and Infection

35.1K 次观看.  University Medical Centre Utrecht. Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

视频: Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

的高分辨电子显微<em&gt;幽门螺杆菌</em&gt;恰IV型分泌系统的霹雳产于铁可用性的条件变

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

马达加斯加嘘声蟑螂作为非哺乳动物的替代动物模型来研究毒力、致病机制和药效

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

幽门螺杆菌感染与胃病理的小鼠模型

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

直立活体显微术制备小鼠颌下腺涎腺

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

功能金报告系统测量特定分枝杆菌误调速率

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...