这项工作是支持的加拿大卫生研究院（MSH - 95368，澳门币89773和MOP - 106623，以锆石）和加拿大创新基金会。 SCM是一个胃肠病学博士后CIHR /加拿大协会的收件人。锆石是一种新CIHR调查。

1。传播鞭muris蛋<ol><li>要生成新的批次的鞭muris蛋，感染20-30免疫缺陷小鼠（如NOD.Cg Prkdc SCID IL2RG tm1Wjl / SZJ（NSG）或129S6/SvEvTac-Rag2 tm1Fwa（RAG2 - / - ））或遗传易感性小鼠（如AKR / J）6-8周约300鞭灌胃muris鸡蛋。 </li><li> CO 2窒息牺牲后32-35天的小鼠。 </li><li>揭露鼠标的腹侧方和湿用70％乙醇的腹部。 </li><li>使用镊子把握腹部皮肤，使一个小切口，用剪刀。切开皮肤暴露在腹腔内容。 </li><li>确定盲肠和大肠近端，并删除它们。 </li><li>放置在一个10厘米的培养皿贝科改良Eagle培养基（DMEM）含500 U / ml青霉素和500微克/毫升链霉素（P / S）的菜的盲肠和大肠近端。切盲肠公开揭露管腔和蠕虫。使用钳地方盲肠在一个烧杯中的磷酸盐缓冲液（PBS），轻轻摇动，以除去粪便。 </li><li>返回盲肠培养皿和使用顺利弯钳（Roboz RS - 5047），轻轻拔出蠕虫和6以及含有5毫升的DMEM + P / S板以及</li><li>重复此过程为所有的老鼠。 </li><li>特百惠容器用湿纸巾保持孵育湿度在37 ° C时4小时，6孔板。 </li><li> 4小时后删除蠕虫使用光滑的弯钳，他们分成2新井的6孔板含5毫升的DMEM + P / S收获在15毫升猎鹰管原作的内容和地点。转速3000 RPM，并删除和储备上清管4小时抗原（可用于细胞restimulation后用PBS透析，典型的蛋白产量是10-20毫克每20只小鼠）和重悬卵灭菌自来水中含有颗粒。 </li><li>将6孔板，37℃过夜。移走的蠕虫。收获成猎鹰管，并在5分钟3000转的旋转井的内容。一夜之间抗原（可用于鞭银的特定抗体的同型酶联免疫吸附后用PBS透析，典型的蛋白产量是10-20毫克每20只小鼠）和重悬卵含在灭菌自来水沉淀取出上清液和储备。 </li><li>结合4小时和隔夜的鸡蛋和鸡蛋通过70μm滤网过滤，以消除任何剩余的蠕虫病毒，然后转移到75厘米的2烧瓶。保持在一个黑暗的地方所涵盖的6个星期在室温（RT）在铝箔的烧瓶。 </li><li>蒸压自来水洗净的鸡蛋和50可行的鸡蛋在50μL重悬。要做到这一点，离心5分钟〜50毫升无菌自来水悬浮在3000 RPM的鸡蛋。在玻片上，取出50μL鸡蛋和地点。使用40倍的放大倍率数在50μL可行的鸡胚。稀释50μL给50个可行的鸡蛋的蛋。对于可行鸡蛋的图像，请参阅研究科珀菲尔德16。 </li></ol> 2。 鞭muris蛋（200粒卵/鼠标）的实验小鼠的急性感染<ol><li>彻底悬浮鸡蛋和删除足够的量来治疗小鼠的数量× 2（如10只小鼠，取200μLx 10只× 2 = 4鸡蛋MLS），并放置在一个14毫升的管理单元帽筒。 </li><li>反转14毫升的管理单元帽筒组合。到一个适当大小的喂养针1毫升注射器（小鼠重25克，我们用18号1 1 / 2“针）200μL的鸡蛋，一方面颈背鼠标和使用，另一方面管理灌胃鸡蛋。 </li><li>等待21天。 </li></ol> 3。实验收获<ol><li>第21天牺牲小鼠使用的CO 2。 </li><li>从小鼠心脏穿刺收集血液，在非肝素的注射器，并储存在4 ° C翌日，分离血清于-20 ° C和冻结血清可用于鞭特定的IgG和IgE的检测采用ELISA法。 </li><li>揭露鼠标的腹侧方和湿用70％乙醇的腹部。 </li><li>使用镊子把握腹部皮肤，使一个小切口，用剪刀。切开皮肤和底层膜暴露腹腔内容。 </li><li>确定小肠，盲肠和结肠。抓住盲肠与钳拉腹腔。用另一双钳推到正确的暴露肠系膜淋巴结（mLNs）小肠。删除mLNs小心，不要削减进入肠道，并在15毫升含2 MLS的媒体猎鹰。如果需要，这些都可以处理，并在体外细胞因子的分析为72小时（4 × 6 /毫升在1微克/毫升αCD3/CD28或50μg/ml4H鞭银在24孔板的1毫升）restimulated ELISA和细胞内f低流式细胞仪。 </li><li>切盲肠和结肠。 </li><li>使用镊子，推动从远端结肠和地方在2毫升爱思进管粪粒。冻结在-20 ° C。粪粒，可抵抗分子β（RELM -β）表达的免疫印迹分析。 </li><li>取出盲肠（约0.5厘米）的提示。要删除，粪便举行钳盲肠提示和冲洗，用PBS在培养皿中使用3毫升的注射器和22号针头。在新鲜的4％多聚甲醛5毫升猎鹰捕捉帽筒的地方。保存在4 ° C。这些都可以石蜡包埋，切片和组织学分析的幻灯片上。 </li><li>取下一块的RNA（〜1厘米）的近端结肠。用500μlRNAlater样品放入2毫升爱思进管。保存在4 ° C。这些样品可通过定量PCR分析mRNA表达。 </li><li>放置于-20 ° C在一个标签的培养皿中，并冻结其余的盲肠</li></ol> 4。在盲肠鞭muris蠕虫枚举<ol><li>从冰箱放置在培养皿〜5毫升的水盘取出盲肠。 </li><li>用剪刀剖开盲肠暴露管腔。 </li><li>与钳举行的盲肠和大力摇晃盲肠删除大部分粪便。 </li><li>盲肠转移到另一个培养皿中含有水和使用光滑的弯钳轻轻刮去IECS的根本组织。 </li><li>盲肠转移到一个新的培养皿中含有水，并积极与钳刮余下的组织，翻录成小块的组织。 </li><li>使用解剖显微镜计数所有的蠕虫在所有的培养皿3。正如你发现一种蠕虫病毒删除从盘，以确保相同的蠕虫，不计两次。如果蠕虫被损坏（即一半打破）只计算厚或薄的两端，以获得准确计数。 </li></ol> 5。代表性的成果 鞭muris感染模型使研究人员能够量化寄生虫负担和全身免疫反应，以及病理检查远端盲肠内的炎症反应。当收获盲肠腔暴露出来， 鞭虫蠕虫可以枚举使用解剖显微镜（图1A - B）。 鞭免疫力参数，还包括抗动物抗体亚型，切换到IgG1的（与Th2型免疫相关），和IgG2a易感动物（与Th1型免疫相关）（图1C ）。杯状细胞特异性蛋白抵抗分子β（RELM -β）的表达与鞭间隙和Western blot分析（图2）检测。盲肠末端的部分，可以与碘酸雪夫（PAS）染色评估杯状细胞的反应和对感染的反应程度的肠道炎症（图3）。 <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="图1"/> 图1。量化的免疫力鞭muris。 （一）抗（B6）小鼠驱逐鞭muris感染后第21天。相比之下，成为慢性感染易感（AKR / J）小鼠与鞭虫 ，导致寄生虫建立。每个条形代表的平均± SEM 4-8动物每组。 （二）蠕虫是使用解剖显微镜计数。顶板显示在隔离蠕虫，底部面板显示蠕虫IECS和粪便的存在。 （三） 鞭特定的IgG2a抗体滴度排泄鞭抗原（O / N银，5微克/涂层钢板是由稀释血清的ELISA（1:2稀释，在1:20稀释开始和结束在1:2560）毫升）。易感AKR / J小鼠鞭特异性血清IgG2a水平较高，特别是较耐B6小鼠。 <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="图2"/> 图2。 鞭muris间隙与杯状RELM -β细胞特异性蛋白7表达 。 RELM -β的生产，可以检测到从鞭虫感染的耐药（B6）的粪粒，但不容易（AKR / J）小鼠免疫印迹分析。每个通道是代表一个单一的动物，（N）=天真，（TM）= 21天鞭虫感染小鼠。 <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="图3"/> 图3。远端盲肠以下鞭虫感染的病理组织学检查。 5-7微米的远端盲肠段与保安局首席助理局长染色。杯状细胞增生和粘液生产明显耐药（B6），但不容易（AKR / J） 小鼠，鞭虫感染。

<ol>
	<li>Cliffe, L. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Trichuris+muris+system:+a+paradigm+of+resistance+and+susceptibility+to+intestinal+nematode+infection.">The Trichuris muris system: a paradigm of resistance and susceptibility to intestinal nematode infection.</a> Adv. Parasitol. 57, 255-307 (2004).</li><li>Else, K. J., Finkelman, F. D., Maliszewski, C. R., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-mediated+regulation+of+chronic+intestinal+helminth+infection.">Cytokine-mediated regulation of chronic intestinal helminth infection.</a> J. Exp. Med. 179, 347-351 (1994).</li><li>Bancroft, A. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1+and+Th2+cells+and+immunity+to+intestinal+helminths.">Th1 and Th2 cells and immunity to intestinal helminths.</a> Chem. Immunol. 71, 192-208 (1998).</li><li>Bancroft, A. J., McKenzie, A. N., Grencis, R. 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+critical+role+for+IL-13+in+resistance+to+intestinal+nematode+infection.">A critical role for IL-13 in resistance to intestinal nematode infection.</a> J. Immunol. 160, 3453-3461 (1998).</li><li>Helmby, H., Takeda, K., Akira, S., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+(IL)-18+promotes+the+development+of+chronic+gastrointestinal+helminth+infection+by+downregulating+IL-13.">Interleukin (IL)-18 promotes the development of chronic gastrointestinal helminth infection by downregulating IL-13.</a> J. Exp. Med. 194, 355-364 (2001).</li><li>Owyang, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+25+regulates+type+2+cytokine-dependent+immunity+and+limits+chronic+inflammation+in+the+gastrointestinal+tract.">Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract.</a> J. Exp. Med. 203, 843-849 (2006).</li><li>Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RELM&#946;/FIZZ2+is+a+goblet+cell-specific+immune-effector+molecule+in+the+gastrointestinal+tract.">RELM&#946;/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 13596-13600 (2004).</li><li>Datta, R. <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+novel+genes+in+intestinal+tissue+that+are+regulated+after+infection+with+an+intestinal+nematode+parasite.">Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite.</a> Infect. Immun. 73, 4025-4033 (2005).</li><li>Cliffe, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+intestinal+epithelial+cell+turnover:+a+new+mechanism+of+parasite+expulsion.">Accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion.</a> Science. 308, 1463-1465 (2005).</li><li>Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+weapons+in+the+war+on+worms:+identification+of+putative+mechanisms+of+immune-mediated+expulsion+of+gastrointestinal+nematodes.">New weapons in the war on worms: identification of putative mechanisms of immune-mediated expulsion of gastrointestinal nematodes.</a> Int. J. Parasitol. 36, 723-733 (2006).</li><li>Finkelman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+regulation+of+host+defense+against+parasitic+gastrointestinal+nematodes:+lessons+from+studies+with+rodent+models.">Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models.</a> Annu. Rev. Immunol. 15, 505-533 (1997).</li><li>Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+helminth+infection:+immunopathology+and+resistance+during+intestinal+nematode+infection.">Enteric helminth infection: immunopathology and resistance during intestinal nematode infection.</a> Chem. Immunol. 66, 41-61 (1997).</li><li>Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+intestinal+muscle+contraction+by+interleukin-9+(IL-9)+or+IL-9+neutralization:+correlation+with+worm+expulsion+in+murine+nematode+infections.">Modulation of intestinal muscle contraction by interleukin-9 (IL-9) or IL-9 neutralization: correlation with worm expulsion in murine nematode infections.</a> Infect. Immun. 71, 2430-2438 (2003).</li><li>Khan, W. I., Blennerhasset, P., Ma, C., Matthaei, K. I., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat6+dependent+goblet+cell+hyperplasia+during+intestinal+nematode+infection.">Stat6 dependent goblet cell hyperplasia during intestinal nematode infection.</a> Parasite Immunol. 23, 39-42 (2001).</li><li>Akiho, H., Blennerhassett, P., Deng, Y., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+IL-4,+IL-13,+and+STAT6+in+inflammation-induced+hypercontractility+of+murine+smooth+muscle+cells.">Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 282, 226-2232 (2002).</li><li>Kopper, J. J., Mansfield, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+improved+methods+for+delivery+of+Trichuris+muris+to+the+laboratory+mouse.">Development of improved methods for delivery of Trichuris muris to the laboratory mouse.</a> Parasitol. Res. 107, 1103-1113 (2010).</li><li>Bancroft, A. J., Else, K. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+infection+with+Trichuris+muris+significantly+affects+the+polarization+of+the+CD4+response.">Low-level infection with Trichuris muris significantly affects the polarization of the CD4 response.</a> Eur. J. Immunol. 24, 3113-3118 (1994).</li></ol>

动物实验是由加拿大英属哥伦比亚大学提出的准则和法规的规定执行。

该协议的细节了一个标准的高剂量急性鞭muris感染研究者的要求，可以修改。例如，老鼠可以牺牲和组织在不同的日子里收获。要确定小鼠已成功建立了完整的蠕虫负担，他们可以被牺牲的第14天，在这一点所有小鼠，应随身携带了约200蠕虫的负担。也可被感染小鼠的32天检测到任何蠕虫将达到成熟阶段，并会维持其生命期限的主机。此外，该协议可以适应慢性鞭虫muris感染模型。要?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>动物饲养针（18 × 1 ½“） </td><td>波普尔</td><td> 7912 </td><td></td></tr><tr><td>光滑的弯钳</td><td> Roboz </td><td> RS - 5047 </td><td></td></tr><tr><td> DMEM培养液</td><td> Gibco公司</td><td> 11965 </td><td></td></tr><tr><td> NOD.Cg Prkdc SCID IL2R gtm1Wjl / SZJ（NSG） </td><td>杰克逊实验室</td><td> 005557 </td><td>这些都是我们所用的老鼠，但是，任何免疫缺陷小鼠或敏感品系的工作。 </td></tr><tr><td> RNAlater </td><td> Qiagen公司</td><td> 76104 </td><td></td></tr><tr><td> 2毫升管</td><td>爱思进</td><td> MCT - 200 - C </td><td></td></tr><tr><td> 15毫升管</td><td>鹘</td><td> 352096 </td><td></td></tr><tr><td> 6孔板</td><td>鹘</td><td> 353046 </td><td></td></tr><tr><td>多聚甲醛</td><td>电子显微学</td><td> 15710 </td><td></td></tr><tr><td> α-RELMβ抗体</td><td> PeproTech公司</td><td> 0694270Rb </td><td></td></tr></tbody></table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

鞭muris是一个自然的小鼠的病原体和鞭虫物种感染人类和牲畜 1的生物学和抗原性相似。感染性虫卵经口灌胃，在远端小肠孵化，侵入肠上皮细胞（IECS），该行盲肠和近端结肠隐窝和成熟后的蠕虫释放到环境中的鸡蛋1。这个模型是一个功能强大的工具来检查因素控制的CD4 +辅助性T细胞（TH）的激活，以及在肠上皮细胞的变化。免疫反应的抗近交系C57BL / 6和BALB / C，如发生的特点，是由Th2细胞极化的细胞因子（IL - 4，IL - 5和IL - 13）和驱逐的蠕虫病毒，而Th1相关的细胞因子（IL -12，IL - 18，IFN -γ）促进在遗传易感性的AKR / J 小鼠 2-6的慢性感染。 Th2细胞因子，促进生理肠道微环境的变化，包括IECS的快速周转，杯状细胞的分化，招聘和上皮通透性和平滑肌收缩，所有这一切都已经在蠕虫驱逐7-15牵连的变化。在这里，我们详细为传播鞭muris可以在随后的实验中使用的鸡蛋的的协议。我们还提供了为感染后的分析建议的样品实验收获。总体而言，该协议将提供的基本工具的研究人员，执行鞭muris小鼠感染模型，它可以用来解决有关的问题在胃肠道以及免疫效应功能的IECS钍倾向。

Watch this Scientific Journal Video about Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut at JoVE.com

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

鞭muris感染肠道模型是一种Th2型免疫抗小鼠Th2反应产生保护和易感小鼠产生一种病态的Th1反应。

鞭虫感染muris：在肠道内的2型免疫和炎症模型

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

trichuris-muris-infection-model-type-2-immunity-inflammation

Research

JoVE Journal

Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

16.2K 次观看.  University of British Columbia. 鞭muris感染肠道模型是一种Th2型免疫抗小鼠Th2反应产生保护和易感小鼠产生一种病态的Th1反应。

视频: Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

科研

免疫与感染

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

味觉信号蛋白废除对炎症性肠病小鼠肠道炎症的影响

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

弥散性念珠菌感染模型

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...