The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

所有的研究获得批准，在弗吉尼亚理工大学IACUC的指导方针进行 1.准备R-Spondin1条件培养基从HEK293T-Rspo1细胞系<ol><li> HEK293T-Rspondin1细胞的产生已如前所述10。种子的HEK293T-Rspondin1分泌细胞:5-10％汇合，约8×10 5 -1.7×10 6个细胞，在为T-175烧瓶用40ml 1×Dulbecco氏改良的Eagle培养基（DMEM）+ 10％胎牛血清（ FBS）的作为生长培养基，并在37℃+ 5％CO 2孵育。 注:对于HEK293T-Rspondin1分泌细胞将作为R-spondin1条件培养基生长培养基。 R-spondin1可替代地购买作为在500纳克/毫升的终浓度中使用的重组生长因子。 </li><li>成长HEK293T-Rspondin1细胞七天或直至达到通过亮视野显微镜测定95％汇合。收获40毫升条件培养基，然后到50毫升锥形管中。丢弃HEK293T-Rspondin1分泌细胞的T形175烧瓶中。 </li><li>离心机含有的条件培养基在300×g离心10分钟，在4℃下在管，以沉淀细胞碎片。收集上清液，称为R-spondin1条件培养基，并分装成15ml试管。商店等分在-20℃下短期或-80℃长期储存。 注意:一旦解冻，则R-Spondin1条件培养基可以存储最多1周4℃。 </li></ol> 2.类器官生长培养基的制备和收获小肠隐窝试剂&#160;<ol><li>收获前一天到，放置在冰上冷冻蛋白基质中并在4℃下解冻过夜。高压釜解剖剪刀，镊子和载玻片，将用于为隐窝收获。 </li><li>第二天，开始通过制备40ml含有补充剂和生长因子类器官的生长培养基中加入库存浓度至40ml 1×DM的EM / F-12。媒体补充剂含有:10mM的2- [4-（2-羟乙基）哌嗪-1-基]乙磺酸（HEPES） - 400微升，1×补充谷氨酰胺- 400微升，1×B27无维生素A - 400微升，1×N 2 - 200微升，1毫摩尔N-乙酰半胱氨酸- 40微升，100纳克/毫升间头蛋白- 40微升，50纳克/毫升间的EGF - 4微升和发生在一个37℃的水浴中，直到使用。在水浴中温R-spondin1的15毫升等分至37℃。 </li><li>温暖3无菌24孔板至37℃至少30分钟通过在培养箱中放置。制备每只小鼠1×磷酸盐缓冲盐水（PBS）+ 2mM的乙二胺四乙酸（EDTA）和凉爽50毫升至4℃。准备每只小鼠100毫升1X含10％FBS，冷却至4℃PBS。 </li></ol> 3.收获小家鼠小肠隐窝为化培养1 <ol><li>牺牲一个雄性C57B6 / J鼠标6-12岁周龄标准的啮齿动物食物和水可用募集根据广告机构指引自由采食 。通过CO 2安乐死窒息颈椎脱位。 注意:保持胴体上的冰上，直到收获的组织，并在生物安全柜进行组织收获，以尽量减少污染的风险。 注:男性或女性鼠标可能被用来生成类器官。 </li><li> （可选）刮胡子鼠标在70％乙醇浸入之前删除的皮毛。浸没在70％乙醇鼠标2分钟，四肢脚用鼠标接触电路板的背侧钉扎板。 </li><li>使用预消毒解剖剪刀和镊子，使鼠标的腹侧部分的中线切口。使在生殖器在进行颅到颈部的底部切口。用镊子横向打开皮肤切口和针皮肤钉扎板以暴露腹膜。 </li><li>使在腹部的腹膜一个中线切口以解剖剪刀和第折Ë腹膜与横向镊子和脚钉扎板，以露出腹部器官。 注:请注意，以避免削减腹部器官。 </li><li>与通过切割连接到胃，并连接到盲肠远端结小肠的近端结解剖镊子和剪刀除去从腹腔小肠。放置小肠中含有10ml冰冷的1×PBS中的无菌培养皿。 </li><li>冲洗载小肠用冰冷1×PBS中使用1毫升吸移管的管腔内的内容。直到小肠的腔中的杂物被除去重复此步骤。 </li><li>切小肠解剖剪刀成3-4大致相等的长度条和纵向奠定一个新的无菌培养皿条。对于每个条带，插入小肠的腔内部的解剖剪刀的刀刃，使一个切口的整个长度ÒF中的条。使用镊子横向折叠打开切口，以揭露小肠腔内。 </li><li>使用无菌载玻片轻轻刮去小肠的腔表面上的绒毛。切小肠进入1-2厘米长条与解剖剪刀且这些条带转移到含有10ml冰冷的1×PBS中的50ml锥形管中。 </li><li>通过反转50ml锥形管轻轻混匀的内容，并允许组织内容沉降到50ml锥形管的底部。吸出1×PBS中并通过用10ml冰冷的1X PBS中洗涤该组织重复此三次。在最后的洗涤留在冰上锥形管含有10ml 1×PBS中，小肠组织片段10分钟。 </li><li>吸出PBS 1X，并添加25毫升1X PBS + 2毫摩尔EDTA的。放置在4℃或在冰桶45分钟在摇摆平台上。虽然组织培养，标签6的15毫升锥形管"分数＃1-6"。 </li><li&gt;温育后，允许组织内容沉降到管和抽吸1×PBS + 2mM EDTA的的底部。加入10 mL 1×PBS中+ 10％FBS的并用手剧烈摇动该管的10倍。 </li><li>允许组织内容沉降到管底部。除去并将上清液转移到标有"级分1"管15毫升锥形。重复1×PBS + 10％FBS的振荡工序（3.11）的五倍，并且为了传送上清液内容至适当标记的馏分管。 </li><li>离心在125×g离心5分钟。吸出上清，重悬粒料在5ml预热1×DMEM / F-12，用无生长因子加入。 </li><li>离心在78×g离心2分钟。抽吸将4ml上清并在1ml剩余介质的悬浮每个粒料。 </li><li>从每一级分移除20个ml和添加到载玻片。隐窝可视化和碎片光显微镜下，并确定哪个组合分数，并相应池实现的最大佩尔切隐窝碎片比ntage。 注:分数2-6产生被镀隐窝的比例最大。到池的部分是最常见的有4分和5分与6分3分。 </li><li>在125 XG待镀5分钟离心馏分。吸上清留下50-100微升其余颗粒之上。 </li><li>保持在冰上管和1ml蛋白基质添加到合并的级分。吸取上下慢慢地防止除气泡。 </li><li>从37℃培养箱除去预热24孔板，加入50μl的蛋白基质/隐窝悬浮液在每个中间良好。引晶的板转移到37℃培养箱中10分钟，以使蛋白基质滴固化。 </li><li>加入450微升事先准备好的器官样生长介质+ R-spondin1的50微升空调每孔媒体。在37℃+ 5％CO 2的过夜孵育板。 </li><li>加入50-100微升R-spondin1空调中介的每天每一个好。每个第 3天更换整个器官样生长介质与新鲜烹制的类器官的生长介质450微升/步骤2.2很好的描述。此外50-100添加微升R-spondin1条件培养基，每孔。 注:该蛋白基质降保持其完整性一个星期，但后7天进行传代类器官。 </li></ol> 4.传代每类器官第 7天<ol><li>在冰上解冻过夜蛋白基质，在4℃下传代的前一天。温暖的无菌24孔板至37℃至少30分钟。准备在步骤2.2中所述类器官的生长培养基，并保持在37℃直到使用。 </li><li>从孵化器中取出类器官板展开板传代冰。从3-4井10毫升吸管吸生长介质启动。 注意:保持在10毫升吸管的送气媒体，因为这将被用于去除在下一步骤中的蛋白基质下降。 </li><li>在如盗版阱，吸管上下以撞出从板的蛋白基质下降。用10毫升吸管的尖端小心刮擦是在撞出蛋白基质降很有帮助。传送内容到15毫升锥形管中。 </li><li>孵育15毫升在冰上锥形管10分钟，并离心在125 xg离心在4℃下5分钟。在锥形管的底部观察到白色沉淀。轻轻吸并丢弃类器官粒料离开100-200微升粒料以上上述大部分上清液/旧类器官生长培养基。 </li><li>通过使用连接到1ml注射器23号针头拆散类器官隐窝。吸并弹出10-15倍，以解离隐窝。减少空气气泡的形成由于过度移液。 </li><li>悬浮组织体于500μl蛋白基质和添加每孔50μl的在一个新的预温热24孔板中。添加预先制备的类器官的生长培养基的450微升+50微升的R-spondin1的每口井条件培养基。孵育在37℃下板过夜。 </li></ol> 5.电镀类器官在14日的模式识别受体刺激与和的PAMP 李斯特菌基因表达分析<ol><li>两天来挑战，连胜了之前L.单核细胞上的BHI琼脂平板上。 </li><li>次日挑L.单核细胞集落，并在37℃下过夜生长在10ml BHI肉汤以200rpm振荡。 </li><li>在冰上解冻过夜蛋白基质，在4℃下类器官挑战的前一天。温暖的无菌24孔板至37℃至少30分钟。准备在步骤2.2中所述类器官的生长培养基，并保持在37℃直到使用。 </li><li>从孵化器中取出化培养，并开始组织体传代。 </li><li>从3-4孔中吸出介质并保持吸气介质在10毫升pi​​pete因为这将被用于去除蛋白质矩阵降。吸取和下来以撞出从板的蛋白基质下降。用10毫升吸管的尖端小心刮擦是在撞出蛋白基质降很有帮助。传送内容到15毫升锥形管中。 </li><li>孵育15毫升在冰上锥形管10分钟。离心机在125×g离心在4℃下10分钟。在锥形管的底部观察到白色沉淀。轻轻吸出上清液留下约100微升残留上清落后。 </li><li>重悬在5ml冰冷的1×PBS中的类器官沉淀并除去一个10微升等分试样用于计数。的10微升等分试样加入到血细胞计数器的中间无玻璃盖玻片。轻轻覆盖在下降顶部的玻璃盖玻片。 注:血细胞计数将与类器官堵塞如果载玻片没有首先去除。 </li><li>通过光学显微镜计数组织体的总数在每个栅格/血细胞计数仪的整个腔室，并确定类器官浓度:NUM共组织体BER每10微升计数。 </li><li>删除15毫升锥形管，将允许组织体的足够数量的待接种用于测定和离心机该悬浮液在125×g离心10分钟，一个适当的体积。 注:每一个24孔板的40-100组织体之间的范围是足够的用于RNA分析。典型的类器官大小的范围可以从300微米至1000微米的直径。 </li><li>吸出上清液并重新悬浮在500毫升的蛋白质基质的粒料不产生气泡。加入50毫升，每孔类器官/蛋白基质悬浮液在24孔板中。 注:蛋白质基质的用于悬浮组织体将用于治疗的条件和技术重复的数目而变化的量。 </li><li>加入500μl类器官生长培养基（无N-乙酰基-半胱氨酸）到每个孔中并在37℃+ 5％CO 2孵育1小时。 </li><li>准备的PAMP的2倍浓度（ 即鞭毛，脂多糖）和生活L.菌等 。现场测量L的OD 菌和牛排在BHI板计数。以1×10 6菌落形成单位（CFU）/ ml的治疗类器官。 注:OD到CFU的决心可能会有所不同细菌的类型，应该事先进行评估，以刺激类器官。例如，0.6≈1×10 9 CFU / ml的李斯特的OD 菌，而且还应当评估独立地之前的实验。 </li><li>连胜走出L的处理股票的连续稀释菌等 ，以准确地确定治疗的CFU /毫升。作者发现，加入100μl的在BHI琼脂平板1×10 8稀释足以计数菌落以确定准确的CFU /毫升。 </li><li>准备L的高温中被杀死的解决方案通过采取活L的1毫升等分试样菌等在5000 xg离心10分钟， 单核细胞溶液和离心机。吸出上清液并洗涤将细菌沉淀用1毫升1×PBS中。 <ol><li>离心机L.在5000 xg离心10分钟， 单核细胞和在1毫升1×PBS中的悬浮颗粒。热火杀L.菌等中在80-90℃进行1小时加热1.7 15mL的聚丙烯管中。文化热的100微升等分打死L.在BHI琼脂平板上的单核细胞 ，以确认没有活的细菌依然存在。 </li><li>加入500微升适当2X PAMP和/或微生物治疗的500微升驻地媒体在由用户确定指定的孔中。孵育在37℃+ 5％CO 2的24小时。 注意:添加2倍PAMP /微生物治疗的500微升等分试样以500μl的驻地媒体会带来处理浓度以1ml的总体积为1x。 </li></ol></li><li>次日，手动计数L。单核细胞集落为准确测定治疗CFU /毫升。从处理过的组织体板吸媒体和洗井Ť重稀土次用1×PBS。 </li><li>通过酚/氯仿11或根据制造商的说明一个RNA提取试剂盒继续进行RNA提取。使用标准的定量RT-PCR 12评估基因表达。 </li></ol> 6.电镀类器官对PAMP和单增李斯特菌挑战上清液中的蛋白质分析14天<ol><li>激发前两天，启动Caco-2细胞的细胞培养物，接种密度≈1×10 6细胞在用1×DMEM + 20％FBS的T-75烧瓶中，并在37℃+ 5孵育的Caco-2细胞％的CO 2。开始L的细菌培养上BHI板菌和在细菌培养箱中于37℃孵育板。 </li><li>次日挑L.单核细胞集落，并在37℃下生长在10ml BHI肉汤过夜。在冰上解冻过夜蛋白基质，在4℃下类器官挑战的前一天。 </li><li>第二天温暖的无菌96孔黑色壁板到37℃，至少30分钟。制备类器官的生长培养基没有在步骤2.2中所述，并保持在37℃直到使用N-乙酰基半胱氨酸。从孵化器中取出化培养，并开始组织体传代。 注意:典型类器官大小可以在直径范围从300微米到1000微米。 </li><li>从3-4孔中吸出介质并保持吸气介质在10毫升pi​​pete因为这将被用于去除蛋白质矩阵降。用移液管重悬以逐出板蛋白质矩阵降。用10毫升吸管轻轻地刮取是撞出蛋白基质下降有帮助。传送内容到15毫升锥形管中。 </li><li>孵育15毫升在冰上锥形管10分钟。离心机在125×g离心在4℃下10分钟。在锥形管的底部观察到白色沉淀。轻轻吸出上清液留下100μl的剩余上清液后面。 </li><li>悬浮颗粒类器官在冰冷的5毫升1X PBS和取出10微升等分用于计数。的10微升等分试样加入到血细胞计数器的中间，并且轻轻覆盖在玻璃盖玻片。 注:血细胞计数将与类器官堵塞如果载玻片没有首先去除。 </li><li>通过光学显微镜计数组织体的总数在血细胞计数器/整个腔室的每一个网格和确定类器官浓度:每10ml计数总组织体的数量。 </li><li>删除15毫升锥形管，将允许组织体的足够数量的待接种用于测定和离心机该悬浮液在125×g离心10分钟，一个适当的体积。 注:蛋白质基质的用于悬浮组织体将用于治疗的条件和技术复制量而变化的量。作者发现，40-100类器官是足够的分泌蛋白分析。 </li><li>吸出上清液并重新悬浮于500μl蛋白基质的颗粒/孔，而不会产生气泡。 注:蛋白质基质的用于悬浮组织体将用于治疗的条件和技术复制量而变化的量。 </li><li>离开至少两列空的96孔黑壁组织培养板中，因为这些将作为井用Caco-2细胞对标准曲线的生成晶种。加入50μl蛋白基质+类器官悬浮液的每孔到预先加温96孔黑壁组织培养板中。储存在37℃的板10分钟以容许蛋白质基质固化。 </li><li>加入100微升类器官生长培养基（无N-乙酰基-半胱氨酸）到每个孔中并在37℃+ 5％CO 2孵育1小时。 </li><li>准备的PAMP的2倍浓度和生活L.菌等。加入100毫升每孔每一个给定处理条件，并培育24小时。 </li><li>翌日板的Caco-2细胞中的标准曲线孔中。通过加热无菌1X PBS，0.25％胰蛋白酶和1x DMEM + 20％FBS T开头的Ø37℃。 </li><li>除去含有Caco-2细胞在T-75烧瓶中，并吸出细胞培养基。清洗细胞，用10ml 1×PBS中。吸出1×PBS中，加2ml的0.25％胰蛋白酶和地点的T-75烧瓶中37℃孵化15分钟。 </li><li>可视化在光学显微镜下的烧瓶以确保细胞脱离然后8毫升1×DMEM + 20％FBS中的添加到分离的Caco-2细胞并转移到15毫升锥形管中。删除10微升等分试样并使用血细胞计数器通过光学显微镜计数细胞。 注:60,000个细胞/孔工作良好和稀释的上部的细胞数可以用于产生标准曲线的情况下进行向下至2,500个细胞/孔。 </li><li>悬浮Caco-2细胞中的蛋白质基质和添加每孔50微升至适当的孔中。允许蛋白质基质在37℃下固化为Caco-2细胞。 注:在Caco-2细胞的生长培养基不需要以下蛋白基质凝固无以复加。 </li><li>以下24个小时的治疗时间为类器官检测，吸并保存类器官细胞的上清液媒体和保持在冰上。用1×PBS洗涤各孔三次。加入100微升100％甲醇中以每孔包括的Caco-2标准曲线孔中，并在4℃下或在冰上孵育20-30分钟来固定类器官。 注:不要求的Caco-2标准曲线孔用1×PBS洗涤，因为它们可以有甲醇直接添加。 </li><li>以下的甲醇孵育，用冰冷的1×PBS洗涤各孔三次。储存在4℃的板长达1-2周。 </li><li>在1微克的浓度加入核染色染料/毫升每孔，用铝箔覆盖板，并在4℃孵育过夜。 </li><li>用96孔板读数器来测量核染色染料的激发/发射（在350nm / 461nm分别）和正常化每类器官以及到Caco-2细胞的标准曲线。 </li><li>进行到下游测定法，诸如酶联免疫吸附<sup&gt; 12，细胞培养物上清液正火细胞数目。 </li></ol>

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The authors declare that they have no competing financial interests.

肠组织体的培养和维护是可以通过用足够的组织培养技术的任何个人掌握的过程。有在传代相比，在更传统的单层生长的细胞时微妙之处，但这些微妙之处不难克服。该方法的关键步骤涉及能够对组织体生长到足够高的密度以获得最佳播种。作为通常可以用细胞系取得了较大的接种密度是不实际的实验，必须缩小与类器官。当有多个处理组，这就变得尤为明显。 该协议的目的是提供一个步骤一步方法研究与各种不同的细菌的肠上皮的宿主 - 病原体相互作用， 病毒和真菌病原体，以及地址的困难使用该系统相对于归一化。报告是可用的describË肠组织体与细菌病原沙门氏菌的相互作用，但测量分泌的细胞因子13时没有解决标准化方法。 有由不同的方法正火类器官培养物时遇到一些困难。通过二喹啉酸法正常化分泌蛋白 （BCA）是一种选择;然而，所需的类器官培养的生长组分（N 2和/或维生素B27）与BCA测定干扰通过细胞生存力（未示出数据）正火，如修饰的MTT法已描述14。然而，这将改变组织体的线粒体代谢活性将通过这种技术，因为MTT引入正常化不精确方法的处理是基于由线粒体脱氢酶15的作用降低。它也有必要从介质除去N-乙酰基半胱氨酸（NAC），而不是在如果立法院通过MTT技术标准化是理想的，但如果有细菌病原体如NAC治疗可以抑制细菌的生长16。 这种技术的优点是，分泌细胞因子和蛋白质的产品类器官，现在可以对Caco-2细胞的标准曲线标准化。该技术的限制是正常化是相关因为未转化的上皮组织体的核染色是针对结肠癌细胞系的核染色正常化。作者发现，与核甲醇和染色细胞核固 染色染料是针对Caco-2细胞的标准曲线的有效正常化技术。虽然这结肠癌细胞系的生长特性不完全模仿肠组织体的生长特性，用核染色剂和测量平均荧光强度是正常化对细胞总数的好策略。

的能力，以收集和小肠，结肠，胰腺，肝脏和脑培养初级组织体已经描述并有密切关系理解一个多种生理学代表现象为组织生物学1-5令人兴奋的进展。描述小肠类器官培养和维护的方法，首次报道了佐藤等人出来汉斯Clevers 1的实验室。在此之前的方法，收获和证明原肠上皮细胞的培养在限于并在维持上皮细胞的生长是无效的。方法包括通过用酶，例如胶原酶和分散酶，这将最终导致混合初级成纤维细胞6的生长培养组织的离解。这些条件也将时间在维持上皮细胞培养物的限制。最小的无上皮细胞小生境将形成，因为上皮细胞会进入细胞凋亡，由于缺乏适当的生长因子或接触丧失完整性，称为anokis 7。所述3D-类器官培养系统的出现提供了含有肠细胞类型的光谱中持续培养1培养原肠细胞的方法。这些上皮组织体具有以上的细胞系是它们是由多种分化的细胞的优点，并更好地模仿它们从体内 8衍生的器官。该过程最终"生长在培养皿迷你肠"已被证明是用于评估肠上皮细胞在不同刺激的反应的有价值的工具。调查主肠细胞微生物病原体相关的分子模式（PAMP）的相互作用是相关的免疫学领域，因为这些分子模式可以调节从主机和微生物9多样的响应。不仅可以调查，现在寻求与鼠标类器官这些互动，但他们可以从人类以及2中培养。该技术具有显着改变的个性化医学的潜力，很容易让人猜测的进步，这种技术将在不久的将来成为可能。 该方法的总的目标是提供一种用于培养，扩展的协议，和治疗肠组织体与各种刺激。这种刺激可以最终范围从疫苗，细菌的PAMP，活的病原体，胃肠（GI）和癌症治疗。小鼠肠道组织体的分离和培养是改编自佐藤等人虽然有从原来的方法略有偏差，最终产品是化培养按照本协议时仍然实现。此方法集中在与非同质细胞结构，其必须基于小区n导电的测定时，可以考虑到工作时，描述为合适正常化的适当技术红棕色。

<table>
	<tbody>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11050</td>
			<td>(Section 1,3,6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Centrifuge</td>
			<td>Thermo</td>
			<td></td>
			<td>(Section 1,3)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GE Healthcare</td>
			<td>Sh30243.01</td>
			<td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td>
		</tr>
		<tr>
			<td>HEK293T-Rspondin1 secreting cell line</td>
			<td></td>
			<td></td>
			<td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&#160;</td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>(Section 1) Or equivalent brand</td>
		</tr>
		<tr>
			<td>T-175 Flask</td>
			<td>Corning</td>
			<td>431079</td>
			<td>(Section 1) Or equivalent brand&#160;</td>
		</tr>
		<tr>
			<td>Protein Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&#160;</td>
		</tr>
		<tr>
			<td>HyClone Dulbecco&#39;s (DPBS)</td>
			<td>GE Healthcare</td>
			<td>SH30264.01</td>
			<td>(Section 2,3)</td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td>(Section 2,3) Advanced DMEM/F12</td>
		</tr>
		<tr>
			<td>Corning 24 Well TC Plates</td>
			<td>Corning</td>
			<td>3524</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>N2 Supplement 100x&#160;</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>B27 without vitamin A 50x&#160;</td>
			<td>Life Technologies</td>
			<td>12587-010&#160;</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Glutamine Supplement (Glutamax)</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12&#160;</td>
		</tr>
		<tr>
			<td>10ml Serological Pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>(Section 2) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>(Section 2) Stock = 100 mg/ml</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>(Section 2) Stock = 1M</td>
		</tr>
		<tr>
			<td>Recombinant Mouse EGF</td>
			<td>Biolegend</td>
			<td>585608</td>
			<td>(Section 2) Stock = 500 mg/ml</td>
		</tr>
		<tr>
			<td>Rocker Variable</td>
			<td>Bioexpres</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting tweezers</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>25 ml Serological Pipet</td>
			<td>Falcon</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SLBB9821</td>
			<td>(Section 3) 0.5M or alternative TC grade EDTA</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>(Section 3) Or equal sized TC dish</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>(Section 4)</td>
		</tr>
		<tr>
			<td>Precision Glide Needle</td>
			<td>Becton Dickinson</td>
			<td>305120</td>
			<td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td>
		</tr>
		<tr>
			<td>Flagellin from Bacillus subtilis</td>
			<td>Invivogen</td>
			<td>tlrl-bsfla&#160;</td>
			<td>(Section 5,6)</td>
		</tr>
		<tr>
			<td>Listeria monocytogenes</td>
			<td>ATCC</td>
			<td>19115</td>
			<td>(Section 5,6)&#160;(Murray&#160;et&#160;al.)&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td>(Section 5,6)Or equivalent brand</td>
		</tr>
		<tr>
			<td>BBL Brain Heart Infusion Agar</td>
			<td>Becton Dickinson</td>
			<td>211065</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>Becton Dickinson</td>
			<td>237500</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Caco-2</td>
			<td>ATCC</td>
			<td>HTB-37</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>gibco</td>
			<td>25200056</td>
			<td>(section 6)</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecuar Devices</td>
			<td></td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>96 Well Assay Plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td>(Section 6) Black Plate, Clear Bottom TC treated</td>
		</tr>
		<tr>
			<td>Nuclear Staining Dye</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td>(section 6) Hoechst 33342</td>
		</tr>
		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td>(Section 6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td>(Section1,3) Or equivalent brand</td>
		</tr>
		<tr>
			<td>1.7 ml polypropylene tube</td>
			<td>Bioexpress</td>
			<td>C-3262-1</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>Quick-RNA MiniPrep</td>
			<td>Zymo Research</td>
			<td>R1054</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>TNF-alpha&#160;</td>
			<td>Applied Biosystems</td>
			<td>Mm 00443260_g1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-6&#160;</td>
			<td>Applied Biosystems</td>
			<td>(Mm 00446190_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-1beta</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434228_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-18</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434225_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>18s</td>
			<td>Applied Biosystems</td>
			<td>Hs 99999901_s1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexus gradient Mastercycler</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Universal PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1 Protein</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>500 ng/ml</td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C7559</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids

主肠组织体是具有显著影响粘膜免疫学领域的潜力的一个有价值的模型系统。然而，的类器官生长特性的复杂性进行了调查显著警告。具体而言，每个单独的器官样的生长模式是高度可变的，并创建在培养的上皮细胞的异质群体。有了这样的警告，共同组织培养做法不能简单应用到类器官系统，由于蜂窝结构的复杂性。计数和电镀对细胞数，这是单独分离的细胞，如细胞系共同仅基于，不是组织体的可靠方法，除非被施加一些正常化技术。正火总蛋白含量因驻地蛋白基质制成复合物。在细胞数量，形状和细胞类型而言，这些特征应在评估分泌CON时加以考虑从类器官质量帐篷。该协议已经产生勾勒出一个简单的程序，以文化和治疗小肠类器官与病原微生物和病原相关分子模式（PAMP）。它还强调当蛋白质分析被这样的挑战之后进行应该应用的标准化技术。

按照此协议，培养肠类器官时，球体特征形类器官会收获后出现。加入R-spondin1条件培养基的每日将启动类器官的生长和出芽。组织体的生长显示于图 1A - F，且代表肠组织体上天1，2，4，5，6和第14天图1F表示在第14天组织体的非均匀生长特性。 一旦组织体生长到足够数量的，可以将它们再接种，用各种的PAMP和/或微生物攻击。可以通过标准技术进行表达分析。这被表示在图2A-C表示其中进行了分析之后一个炎性细胞因子IL-18，IL-6和TNFα的mRNA表达具有清热组织体24小时挑战打死L.单核细胞增生和PAMP鞭毛蛋白。用于评估mRNA表达的理由细胞因子IL-18，IL-6和TNFα的是，这些是良好的候选响应于致病挑战由上皮细胞被调制，而这些炎性细胞因子的调制将表明该技术的有效性。 图3A - ç演示肠类器官用在与驻地蛋白基质碎片最小的背景染色固定核染色染料的相对核染色。可应用于固定和染色的这种方法正常化测定法，其中将占在每个不同的细胞数好。产生镀在蛋白基质Caco-2细胞的连续稀释的标准曲线时，这是在图4中明显看出，然后固定并用核染色染色染料。 0.89的R 2值表明之间细胞数呈线性关系，平均荧光强度，线性方程可以用于标准化组织体细胞数。在图4所示的标准曲线开始达到饱和极限之外，每孔30,000个细胞。细胞以上，每孔30,000个细胞的滴定已在图4中被示为包括核染色染料的饱和范围。正常化的增加的准确度将与反映了核染色的染料的线性范围达到饱和极限之前的细胞数目而获得。 <img alt="图1" src="/files/ftp_upload/54033/54033fig1.jpg" /> 图1:对于下列隔离小鼠小肠衍生组织体生长的小肠组织体生长的时间过程。 （A）第1天（B）第2天（C）第4天（D），5日（E）6天（F）14日图像代表每一天类器官的增长。比例尺等于200微米A的左图像中，B，C，D和E.比例尺为A，B，C，D和E.比例尺右图像中等于100微米等于1000微米和200微米对于适用于F左，右图像，分别为。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54033/54033fig2.jpg" /> 图2:按照24小时PAMP挑战炎性细胞因子mRNA表达的挑战与和的PAMP热灭活李斯特菌 24小时野生型肠组织体相对表达（A - C）表示折我的变化。<html> nflammatory细胞因子IL-18，IL-6和TNFα的分别。误差线表示标准偏差（SD） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54033/54033fig3.jpg" /> 图3:组织体的规格化相对荧光染色与以下固定组织体细胞核染色。 （ 一 ）组织体明场。 （B）与核染色染料类器官的荧光染色。 （C）明场和荧光染色的合并图像。比例尺等于400微米的所有图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "&gt; <img alt="图4" src="/files/ftp_upload/54033/54033fig4.jpg" /> 图4:从Caco-2细胞产生的标准曲线从一式三份孔中产生的标准曲线。细胞种植了范围开始，每孔60,000个细胞稀释到每孔2500细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54033/54033fig5.jpg" /> 图5:该议定书图的概述图1-顶部面板示出收获的R-spondin1条件培养基。图2，中间面板显示协议收获和培养小鼠小肠类器官的各个方面。图3，底部面板显示:（A）的第14天培养类器官的电镀。用的PAMP / L（B）挑战菌，并留下未占用井以产生标准曲线（C）继PAMP挑战，Caco-2细胞中的先前未被占据的孔电镀来产生标准曲线。 （D）继固定和另外一个核染色染料，测量井和规范与标准曲线的激发/发射。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

这里，收获，维护和治疗与病原体相关的分子模式（PAMP），并李斯特菌小鼠小肠组织体的协议被描述，以及强调基因表达和蛋白质正确规范化技术。

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

13.5K 次观看.  Virginia Maryland College of Veterinary Medicine, Virginia Tech. 这里，收获，维护和治疗与病原体相关的分子模式（PAMP），并李斯特菌小鼠小肠组织体的协议被描述，以及强调基因表达和蛋白质正确规范化技术。 

视频: The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

科研

免疫与感染

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

在抗病毒模式识别受体RIG-I和PKR通过有限蛋白酶消化和Native PAGE监测激活

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

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.

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. ...

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

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

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

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

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

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

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

该<em&gt;离体</em&gt;结肠器官培养及使用抗菌宿主防御研究

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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

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

小鼠小肠免疫系统淋巴细胞的分离

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

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, ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...