This work was supported by grants from the Institute of Science and Technology of the Federal District (ICyTDF, 64/2012 to EO) and the Mexican Council for Science and Technology (Conacyt, 179895 to MS).

1，E.建立和维护阿米巴文化<ol><li>成长axenically（完全免费所有其他污染微生物）1×10 5滋养体阿米巴株HML的:IMSS克隆18在16 x 125毫米培养管聚四氟乙烯内衬螺旋盖（或1×10 6滋养体在一次性的T- 25烧瓶中）和15毫升（或50毫升缇-S-33培养基中的T-25烧瓶中）（缇肉汤补充有3％的钻石维生素混合物，10％热灭活的成年牛血清，0.5国际单位/毫升青霉素和35微克/毫升链霉素）19在培养箱中在37℃下</li><li>在通常的对数生长期在48-96小时的时间间隔（ 图1）通过冷却的培养管中5-10分钟，在冰-水浴中以释放附着在玻璃培养管滋养体收获滋养体。 </li><li>转让文化成锥形管和颠倒几次驱散CELLS。确定细胞数用血细胞计数（纽鲍尔室）和接种物转移到含有新鲜缇-S-33培养基中培养管。 <ol><li>使用低号变形虫更长的潜伏期（〜3×10 5细胞〜5天）和更短期间内有较大的数字（〜1×10 6细胞〜1天）。虽然计数接种是可取的，建立文化变得可预测的，这样接种的估计量是可行的。 </li><li>滴定滋养体的量，以优化细胞数为每一个实验。 </li><li>保持平行重复的文化有一个备份，以防不慎污染或管破损。 </li></ol></li><li>管帽紧紧地和孵化它们在37℃的水平倾斜角度5°。 </li><li>检查文化通过视觉检查定期，因为文化的过快增长可能包含许多裂解细胞。 </li></ol> 2，建立MDCK文化和维护&lt;/ P&gt; <ol><li>生长的MDCK（的Madin Darby狗肾细胞）的I型（高电阻），在一次性的T-75培养瓶中，用10ml补充有青霉素（100国际单位/毫升），链霉素（100毫克/毫升）的DMEM培养基中，胰岛素（0.08 U /毫升）和10％新生小牛在5％CO 2和95％空气的潮湿气氛中，在37℃血清</li><li>要拆分的单元格，检查他们在倒置显微镜。拆分单元格时，他们〜85-100％汇合。 </li><li>使用真空吸气器在无菌罩和无菌的玻璃巴斯德移液管从培养介质中删除。为了避免刮细胞，转瓶倒过来，抽吸介质从底部角落。 </li><li>分配10毫升预热的PBS（140 mM氯化钠，2.7 mM的氯化钾，10毫米的Na 2 HPO 4，1.8 mM的KH 2 PO 4，pH 7.4）中的导入T-75烧瓶（5毫升在T-25烧瓶中的情况下），并轻轻摇动烧瓶以洗涤细胞。真空抽吸去除PBS。彻底清洗是必需的，因为血清抑制胰蛋白酶。 </li><li>分配1.5毫升0.05％胰蛋白酶的导入T-75烧瓶（对于T-25烧瓶中，用0.5ml）中，并轻轻摇动烧瓶以覆盖细胞层，用胰蛋白酶。 </li><li>孵育在孵化5-10分钟（具体时间取决于胰蛋白酶的活性）。 </li><li>检查细胞脱落握住瓶对着光，寻找流动的细胞。细胞脱离（四舍五入细胞）也可使用显微镜检查。通常情况下，细胞释放速度更快的快速，温和的巴掌烧瓶的一侧。 </li><li>加15毫升补充的DMEM对T-75烧瓶（5毫升一T-25烧瓶中）。移液器向上和向下4〜5次重悬细胞。 </li><li>传播的细胞，淡化细胞悬液1:5辅以新鲜的DMEM。在这一点上使用血球细胞数也可确定，而这通常是只有必要的，如果分裂细胞分化成特殊格式的某些测定法。 </li></ol>滋养体的总裂解液3。准备<ol><li>从铜分离滋养体lture管通过冷却所述管在冰 - 水浴中5〜10分钟。转移培养无菌成锥形管中并离心分离，在360×g离心10分钟，在4℃下小心弃去上清液通过倾析，加入冰冷的PBS至沉淀，颠倒试管几次，在360×g离心10分钟，再以分散细胞并离心。重复此步骤两次，以除去缇-S-33介质的所有痕迹。 </li><li>使用血细胞计数器确定滋养体数目。 </li><li>裂解滋养体通过冻融。卡扣冻结滋养体的测定接种物（600000滋养体/毫升）稀释，在冰冷的PBS中，在液氮中1分钟。解冻的样品在4℃和涡大力。重复冻融3次，以完成细胞裂解。 </li><li>激活存在于溶胞产物用0.02％β-巯基乙醇的蛋白酶。 </li></ol> 4，准备滋养体分泌的产品<ol><li>执行步骤3.1至3.2。 </li><li>确定使用血细胞计数器，并应用锥虫蓝排斥试验20细胞数目和生存力在这一点: <ol><li>稀9×10 6个滋养体在3毫升冰冷的PBS和转移细胞到50ml锥形管中。取10微升此悬浮液，加入1微升0.4％台盼蓝储备液pH值7.2。 </li><li>使用纽鲍尔室计数细胞在低倍镜下。 </li><li>计数所有的细胞和分离的蓝色细胞的数量，因为蓝色的细胞已占用的染料，必须予以考虑死。 </li><li>通过将活细胞的数目通过细胞的总数并乘以100计算出的存活细胞的百分比。 </li></ol></li><li>转移的锥形管含有滋养体悬浮到培养箱中在37℃下进行2小时的倾斜水平角度5°。 </li><li>收集的分泌产物，离心管在360×g离心10分钟。使用一次性无菌注射器，仔细收集苏pernatant，避免样品污染的沉淀滋养体。通过使上清液通过0.22μm的醋酸纤维素膜消除任何转移的细胞。激活蛋白酶与0.02％β-巯基乙醇。 </li><li>要放弃假定不需要的污染与死亡细胞释放的分子，重新应用台盼蓝排斥试验细胞活力。如在步骤4.2获得的细胞的存活和总数量应该是相同的。如果不是这样的情况下，所收集的上清液可以不仅含有分泌的蛋白质，应该被丢弃。 </li></ol> MDCK细胞5。与滋养体，滋养体裂解物或分泌产品互动<ol><li>融合培养MDCK细胞单层用活滋养体（1:1的MDCK对阿米巴比），滋养体裂解液（1:2的MDCK对阿米巴比）或分泌的产品（1一MDCK对阿米巴比例:10），在37℃下2分钟和30分钟（ 图2A）。 </li><li>用冰冷的PBS洗涤上皮细胞5次，以消除未结合的分子或滋养体。 </li></ol> 6，样品的制备用于免疫荧光<ol><li>培养MDCK细胞在无菌盖玻片置于24多孔细胞培养皿内。检查在倒置显微镜的细胞，直到它们达到汇合的100％。然后，更换培养基，用1ml新鲜辅以DMEM培养基中，并在板转移到37℃培养箱中放置24小时以上。 </li><li>使用真空吸气器和一个无菌的玻璃巴斯德移液管取出培养基，并加入1毫升温的PBS中，以每个孔中。除去PBS，重复用新鲜PBS洗。小心不要从井与玻璃吸管底部刮细胞。 </li><li>添加滋养体稀释1毫升温PBS的不同情况:现场滋养体（T），滋养体总裂解液（TL）和分泌的产品（SP）。 <ol><li>将培养板在培养箱中培养2或30分钟。 </li><li>准备两个控制条件:命名时1 0分，其中MDCK细胞应该不会孵育E.阿米巴但只用1毫升温的PBS;和其他条件如二抗控制，在这种情况下温育的MDCK带T，TL或SP 2或30分钟，并省略孵化与初级抗体。 </li></ol></li><li>用冷PBS洗上皮细胞五次，以消除未结合的分子或滋养体。 </li><li>固定和透化的细胞用1毫升96％乙醇中30分钟，在-20℃下</li><li>除去乙醇，执行三个快速洗涤用1毫升的PBS在室温下。 </li><li>执行在潮湿的室内下列步骤，以避免样品的干燥: <ol><li>使用任何可用的扁平盒子，可以封闭并且填充有湿纸巾在其上的样品可以被安全地放置。例如，一个空的枪头盒为这个目的很好。 </li><li>内室，均匀地将一个封口膜层和吸管25微升块ING液（0.5％BSA）为每个盖玻片。 </li><li>取盖玻片出井用细镊子，小心地从边缘除去多余的液体用滤纸，把盖玻片到含有封闭液（0.5％BSA）中与细胞面临的封闭溶液的下降。孵育样品1小时，在室温下进行。 </li></ol></li><li>制备初级抗体的混合物在PBS:IgM的鼠抗EhCPADH112（Mα-EhCPADH112; 1:10稀释）和IgG兔抗ZO-1（Pα-ZO-1; 1:100稀释）。 </li><li>将封口膜的全新片在潮湿的盒子和吸管25微升的每个盖玻片抗体的解决方案。 </li><li>从封闭溶液提起盖玻片，晾干任何多余的液体在使用滤纸的边缘，并将其放置在与细胞面临的抗体溶液的液滴。过夜孵育样品在4℃下</li><li>把每个盖玻片到24多井的井（单元侧的顶部），加1毫升的PBS。 <ol><li&gt;洗涤5次，用1ml的PBS中，以去除未结合的分子。 </li></ol></li><li>制备在PBS中的荧光第二抗体的混合物:山羊抗小鼠IgM抗体结合到FITC（1:100稀释）和山羊抗兔IgG抗体结合到TRITC（1:50稀释）。 </li><li>将封口膜的全新片放入潮湿箱和吸管25微升每个盖玻片的二级抗体溶液中。 </li><li>转移样品在6.10和孵育1小时，在室温下在黑暗中以避免荧光染料的褪色。重复步骤6.11.1。 </li><li>对核染色，孵育3分钟，加入200μl0.05mM的DAPI溶液在室温和避光。重复步骤6.11.1。 </li><li>为了保护荧光染料，将盖玻片（细胞面朝下）插入5微升Vecta盾抗淬灭的显微镜玻片安装解决方案。使用指甲油，避免样品干燥密封盖玻片。 </li><li>对于长期储存，保持幻灯片的在-20℃下的显微镜载玻片盒</li><li>通过Z堆叠部分和XZ-平面（ 图2A）分析制剂通过共聚焦显微镜。 </li></ol> 7，培养滋养体与蛋白酶抑制剂或特异性抗体<ol><li>重复步骤3.1至3.2。对于每种实验条件下，培养1×10 5个滋养体（重悬浮于50μlPBS中）与蛋白酶抑制剂（1mM的完整和40微克/毫升的E-64）或抗EhCPADH112（mαEhCPADH112）30微克的单克隆抗体21为20分钟，在4 ℃（ 图2B）。使用这些制剂共培养试验按照步骤8.5中所述。 </li></ol>跨上皮电阻的8。测量（TER） <ol><li>培养MDCK细胞在无菌的transwell透水支持（0.4微米孔径）。在上部隔室中，将100μl的细胞悬浮液，并在下部隔室把600微升补充DMEM培养基。 <ol><li>定期检查的中等水平。新鲜培养基可以根据需要添加。 </li><li>检查在倒置显微镜的细胞，直到它们达到汇合的100％。改变培养基每2-3天。 </li><li>以改善细胞的附着（如需要），加入细胞悬浮液前过夜平衡的transwell小的过滤器，在37℃在DMEM中。 </li></ol></li><li>在乙醇消毒STX2电极在通风橱中通过浸渍，然后在无菌PBS中，每次15分钟，在紫外光下。连接电极的EVOM上皮voltohmmeter并选择电阻模式。 </li><li>从每个Transwell小的上格收集中，汇集他们。取50微升这种混合物介质，并用50μl的滋养体悬浮液（1×10 5滋养体在PBS中），或只用PBS（对照组）相结合。每个Transwell小使用类似的混合。 </li><li>加的混合物，以每Transwell小containin的上部腔室克的MDCK细胞。实验条件包括MDCK细胞而不滋养体（对照），共培养与滋养体或滋养体预温育与蛋白酶抑制剂或mαEhCPADH112。为了消除过滤器提供的阻力，使用转孔孵育唯一媒介。对于每种实验条件下利用至少三个转孔。 </li><li>随即，测量TER通过浸渍STX2电极到每个Transwell小。 <ol><li>确保较长的电极接触的下腔室的底部，而较短的电极覆盖介质在上部隔室（参见图2B）。 </li><li>请务必保持电极每次垂直。这将提高可重复性，显著。避免移动或在倾斜测量电极，因为这会导致数据的变化。 </li></ol></li><li>这种测量应被视为初始TER值。然后，在下面的过程中监控的TER30分钟。 </li><li>来计算最终的TER值，减去的只是过滤器的TER值。以从不同的实验比较结果正常化的每个数据点的初始值，取它们作为100％（ 图2B）。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine+proteases+of+parasitic+organisms.">Cysteine proteases of parasitic organisms.</a> Mol Biochem Parasitol. 120, 1-21 (2002).</li><li>Betanzos, 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=The+EhCPADH112+complex+of+Entamoeba+histolytica+interacts+with+tight+junction+proteins+occludin+and+claudin-1+to+produce+epithelial+damage.">The EhCPADH112 complex of Entamoeba histolytica interacts with tight junction proteins occludin and claudin-1 to produce epithelial damage.</a> PLoS One. 8, (2013).</li><li>Lauwaet, 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=Proteinase+inhibitors+TPCK+and+TLCK+prevent+Entamoeba+histolytica+induced+disturbance+of+tight+junctions+and+microvilli+in+enteric+cell+layers+in+vitro.">Proteinase inhibitors TPCK and TLCK prevent Entamoeba histolytica induced disturbance of tight junctions and microvilli in enteric cell layers in vitro.</a> Int J Parasitol. 34, 785-794 (2004).</li><li>Leroy, 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=Contact-dependent+transfer+of+the+galactose-specific+lectin+of+Entamoeba+histolytica+to+the+lateral+surface+of+enterocytes+in+culture.">Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture.</a> Infect Immun. 63, 4253-4260 (1995).</li><li>Leroy, A., Lauwaet, T., De Bruyne, G., Cornelissen, M., Mareel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entamoeba+histolytica+disturbs+the+tight+junction+complex+in+human+enteric+T84+cell+layers.">Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers.</a> FASEB J. 14, 1139-1146 (2000).</li><li>Leroy, 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=Disturbance+of+tight+junctions+by+Entamoeba+histolytica:+resistant+vertebrate+cell+types+and+incompetent+trophozoites.">Disturbance of tight junctions by Entamoeba histolytica: resistant vertebrate cell types and incompetent trophozoites.</a> Arch Med Res. 31, (2000).</li><li>Lauwaet, 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=Entamoeba+histolytica+trophozoites+transfer+lipophosphopeptidoglycans+to+enteric+cell+layers.">Entamoeba histolytica trophozoites transfer lipophosphopeptidoglycans to enteric cell layers.</a> Int J Parasitol. 34, 549-556 (2004).</li><li>Kissoon-Singh, V., Moreau, F., Trusevych, E., Chadee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entamoeba+histolytica+Exacerbates+Epithelial+Tight+Junction+Permeability+and+Proinflammatory+Responses+in+Muc2(-/-)+Mice.">Entamoeba histolytica Exacerbates Epithelial Tight Junction Permeability and Proinflammatory Responses in Muc2(-/-) Mice.</a> Am J Pathol. 182, 852-865 (2013).</li><li>Lejeune, M., Moreau, F., Chadee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+produced+by+Entamoeba+histolytica+signals+via+EP4+receptor+and+alters+claudin-4+to+increase+ion+permeability+of+tight+junctions.">Prostaglandin E2 produced by Entamoeba histolytica signals via EP4 receptor and alters claudin-4 to increase ion permeability of tight junctions.</a> Am J Pathol. 179, 807-818 (2011).</li><li>Orozco, M. E., Martinez Palomo, A., Gonzalez Robles, A., Guarneros, G., Galindo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+lectin+and+receptor+mediate+the+adhesion+of+E.+histolytica+to+epithelial+cells.+Relation+of+adhesion+to+the+virulence+of+the+strains.">Interactions between lectin and receptor mediate the adhesion of E. histolytica to epithelial cells. Relation of adhesion to the virulence of the strains.</a> Arch Invest Med (Mex). 13 Suppl 3, 159-167 (1982).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+medium+for+the+axenic+cultivation+of+Entamoeba+histolytica+and+other+Entamoeba.">A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.</a> Trans R Soc Trop Med Hyg. 72, 431-432 (1978).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr Protoc Immunol. Appendix 3, (2001).</li><li>Arroyo, R., Orozco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+identification+of+an+Entamoeba+histolytica+adhesin.">Localization and identification of an Entamoeba histolytica adhesin.</a> Mol Biochem Parasitol. 23, 151-158 (1987).</li><li>Diamond, 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=Axenic+cultivation+of+Entamoeba+hitolytica.">Axenic cultivation of Entamoeba hitolytica.</a> Science. 134, 336-337 (1961).</li><li>Diamond, 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=Techniques+of+axenic+cultivation+of+Entamoeba+histolytica+Schaudinn,+1903+and+E.+histolytica-like+amebae.">Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae.</a> J Parasitol. 54, 1047-1056 (1968).</li><li>Dukes, J. D., Whitley, P., Chalmers, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MDCK+variety+pack:+choosing+the+right+strain.">The MDCK variety pack: choosing the right strain.</a> BMC Cell Biol. 12, (2011).</li><li>Espinosa-Cantellano, M., Martinez-Palomo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+intestinal+amebiasis:+from+molecules+to+disease.">Pathogenesis of intestinal amebiasis: from molecules to disease.</a> Clin Microbiol Rev. 13, 318-331 (2000).</li><li>Martinez-Palomo, 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=Structural+bases+of+the+cytolytic+mechanisms+of+Entamoeba+histolytica.">Structural bases of the cytolytic mechanisms of Entamoeba histolytica.</a> J Protozool. 32, 166-175 (1985).</li><li>Martinez-Lopez, C., 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+EhADH112+recombinant+polypeptide+inhibits+cell+destruction+and+liver+abscess+formation+by+Entamoeba+histolytica+trophozoites.">The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites.</a> Cell Microbiol. 6, 367-376 (2004).</li><li>Ocadiz-Ruiz, 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=Effect+of+the+silencing+of+the+Ehcp112+gene+on+the+in+vitro+virulence+of+Entamoeba+histolytica.">Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica.</a> Parasit Vectors. 6, 248 (2013).</li><li>Johnson, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+imaging+techniques+to+studies+of+epithelial+tight+junctions.">Applications of imaging techniques to studies of epithelial tight junctions.</a> Adv Drug Deliv Rev. 57, 111-121 (2005).</li></ol>

The authors have nothing to disclose.

为了研究上皮感染时体外宿主-病原体相互作用的E.组织阿米巴 ，关键的是与两个上皮细胞和滋养体行之有效的培养工作。例如，以前，E.阿米巴培养了通常建立在与细菌或锥虫22,23某些物种的关联。然而，共培养的大肠杆菌阿米巴培养是得不偿失的宿主-病原体相互作用的研究，因为在宿主细胞中观察到的效果不能明确归因于ameobas但可以相当是共培养细胞的效果?...

阿米巴是一种单细胞原生动物负责人阿米巴病，肠道感染引起炎症和腹泻。E.阿米巴感染高达50亿人每年，但只有约10％的感染者制定相关的阿米巴病1，症状。感染发生于对含有大肠杆菌污染的食物或水摄入阿米巴包囊。在肠道内，囊肿产生坚持结肠粘蛋白和增殖2现场滋养体。滋养体通常形成是通过粪便排出体外囊肿。在其他情况下和未知的原因，滋养体打破肠上皮细胞层和侵入皮下组织。在最坏的情况下，他们进入血流而影响其他器官，如肝3。 打破上皮屏障需要保持细胞加入transmembranal上皮结构的破坏。上皮细胞触点由根尖连接复合体组成的紧（TJ）形成和粘合连接（AJ）和桥粒4。最顶端的交界处TJ，因此，它们是由E.冒犯的第一阻挡主机入侵期间组织阿米巴和一些其他病原体。 TJ是由transmembranal粘附受体如claudins，occludin和连接粘附分子（JAM）从事均聚物或嗜异性的相互作用与所述相邻小区的受体。它们是由紧密连接（ZO）系列连接粘附受体与肌动蛋白细胞骨架，以提供进一步的机械强度上皮的支架分子在细胞内的约束。 TJ负责密封肠组织从肠腔，防止过多的水分和溶质渗漏。因此，后TJ由寄生虫破坏，组织被入侵。E.阿米巴分泌一些分子，如:（ⅰ）涉及变形虫的粘附性尔格等细胞5; （ii）在胞吐作用参与杀伤宿主细胞的膜活性的因素，例如被称为离子通道形成肽amoebapores 6,7;及（iii）降解细胞外基质蛋白并介导组织崩解5,8,9蛋白酶。 半胱氨酸蛋白酶EhCP112和粘附分子EhADH112，它们一起组成EhCPADH112复杂两种E.阿米巴毒性蛋白，在TJ 10的拆装发挥了重要作用。现场滋养体，他们的总裂解液和分泌产物诱导的上皮屏障的TJ复杂和功能障碍的分子变化。在这项研究中，它表明，EhCP112和EhADH112与occludin和紧密连接蛋白-1的蛋白质，导致内在化和细胞蛋白的降解相互作用，从而有利于E。通过旁细胞途径阿米巴入口。 我们的数据和那些ØF其他群体11-17强烈建议特定的宿主-病原体相互作用，使寄生虫侵袭的必要性。揭开这些相互作​​用的分子基础是最重要的一个更好的了解阿米巴病的发病机制。 TJ由滋养体，其特征在于，增加细胞旁渗透性选择性的干扰，可以通过在跨上皮电阻下降（TER）来测量。寄生蛋白对宿主上皮细胞的转移可以通过免疫荧光染色和共聚焦激光显微镜，也可以揭示共定位阿米巴毒力因子的上皮交界性标志物，指示可能的直接的相互作用的方法来确定。在这篇文章中，我们将详细介绍上皮细胞和滋养体如何栽培，收获和操纵研究宿主 - 病原体相互作用及其后果。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Entamoeba histolytica HM1:IMSS, Clone A&#160;</td>
			<td>IMSS Hospital, Mexico</td>
			<td>Without/number</td>
			<td>Virulent trophozoites18&#160;</td>
		</tr>
		<tr>
			<td rowspan="8">TYI broth</td>
			<td rowspan="8">Becton, Dickinson and Company 
			Merck 
			Merck 
			Merck 
			J.T. Baker 
			Reproquifin 
			SIGMA-Aldrich 
			SIGMA-Aldrich</td>
			<td rowspan="8">211862 
			K35625437 626 
			21578 
			4873 
			3252-01 
			CAS 50-81-7 
			C7880 
			F-5879</td>
			<td rowspan="8">3.45% BBL Biosate peptone 
			58 mM glucose 
			39 mM NaCl 
			5 mM KH2PO4 
			6.5 mM K2HPO4 
			16.3 mM ascorbic acid 
			8.1 mM L-cysteine 
			&#160;0.1 mM ferric ammonium citrate, adjust pH 6.819</td>
		</tr>
		<tr>
			<td>Bovine serum adult</td>
			<td>Microlab , Labs., Mex.</td>
			<td>SU146</td>
			<td>Use at 10% and inactivated to 56&#176; C for 30 min</td>
		</tr>
		<tr>
			<td>Diamond&#160; vitamin&#160; mixture- Tween 80</td>
			<td>In vitro</td>
			<td>SR-07</td>
			<td>Use at 3%</td>
		</tr>
		<tr>
			<td>Penicillin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>34564SSA IV</td>
			<td>0.5 IU/mL</td>
		</tr>
		<tr>
			<td>Streptomycin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>75757SSA IV</td>
			<td>35 &#181;g/ mL</td>
		</tr>
		<tr>
			<td>Pyrex 15 mL screw cap culture tubes with PTFE lined phenolic caps</td>
			<td>Corning-Pyrex</td>
			<td>9826-16X</td>
			<td>16x125 mm, capacity 15 mL and caps fabricated from special formula resistant to effects of temperature</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6 Well</td>
			<td>Corning-Costar</td>
			<td>3516</td>
			<td>Sterile plates, well diameter 34.8 mm and growth area 9.5 cm2.&#160; Rings on lid prevent cross-contamination</td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430168</td>
			<td>Canted neck flasks</td>
		</tr>
		<tr>
			<td>MDCK (Madin Darby canine kidney) type I</td>
			<td>American Type Culture Collection</td>
			<td>CCL34</td>
			<td>Kidney epithelial cells grown between the 60th and 90th passage</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>12800-017</td>
			<td>Dulbecco&#39;s Modified Eagle Medium with high glucose.</td>
		</tr>
		<tr>
			<td>Neonate Calf Serum</td>
			<td>In vitro</td>
			<td>S-02</td>
			<td>Use at 10%.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin mixture&#160;</td>
			<td>In vitro&#160;</td>
			<td>A-01</td>
			<td>Stock solution 10,000 U/&#181;g/mL</td>
		</tr>
		<tr>
			<td>Insulin&#160;&#160;</td>
			<td>AMSA</td>
			<td>398MJ94SSA IV</td>
			<td>Stock solution 100 IU/mL</td>
		</tr>
		<tr>
			<td>Trypsin solution&#160;</td>
			<td>In vitro</td>
			<td>EN-005</td>
			<td>0.05% enzyme solution without calcium and magnesium</td>
		</tr>
		<tr>
			<td>75cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430720</td>
			<td>Canted neck flasks for trophozoite culture in TYI-S-33 medium</td>
		</tr>
		<tr>
			<td>Transwell permeable supports</td>
			<td>Corning-Costar</td>
			<td>3470</td>
			<td>0.4. &#181;m polyster membrane, 6.5 mm insert in 24 well plate, growth area 0.3 cm2</td>
		</tr>
		<tr>
			<td>24 well cell culture dish&#160;&#160;</td>
			<td>Corning-Costar</td>
			<td>3524</td>
			<td>Clear polystyrene, treated for optimal cell attachment, sterilized by gamma radiation and certified non-pyrogenic</td>
		</tr>
		<tr>
			<td>Complete Mini</td>
			<td>Roche</td>
			<td>11836 153 001</td>
			<td>Protease inhibitor cocktail inhibits a broad spectrum of serine, cysteine and metallo-proteases. Final concentration 1 mM&#160;</td>
		</tr>
		<tr>
			<td>Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64)</td>
			<td>SIGMA-Aldrich</td>
			<td>E3132</td>
			<td>Cystein protease inhibitor, final concentration 40 &#181;g/mL</td>
		</tr>
		<tr>
			<td>p&#945;ZO-1&#160;</td>
			<td>Invitrogen</td>
			<td>402200</td>
			<td>IgG rabbit policlonal&#160; antibody&#160; against&#160; a synthetic peptide in the middle region of the ZO-1 human protein</td>
		</tr>
		<tr>
			<td>m&#945;EhCPADH112</td>
			<td>Homemade antibody</td>
			<td>Without/ Number</td>
			<td>IgM mouse monoclonal antibody&#160; against&#160; 444-601 epitope located at C-terminal of EhCPADH11221,27</td>
		</tr>
		<tr>
			<td>FITC-goat anti-mouse IgM</td>
			<td>Zymed</td>
			<td>62-6811</td>
			<td>Fluorescein isotiocyanate (FITC)-labelled goat anti-mouse secondary&#160; antibody</td>
		</tr>
		<tr>
			<td>TRITC- goat anti-rabbit IgG (H+L)</td>
			<td>Zymed</td>
			<td>816114</td>
			<td>Tetramethyl-rhodamine isothiocyanate (TRITC)-labelled&#160; goat anti-rabbit IgG&#160; secondary antibody.</td>
		</tr>
		<tr>
			<td>STX2 Electrode</td>
			<td>World Precision Instrument&#160;</td>
			<td>102711</td>
			<td>Consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick. Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. For use with EVOM</td>
		</tr>
		<tr>
			<td>EVOM epithelial voltohmmeter</td>
			<td>World Precision Instrument&#160;</td>
			<td>12111</td>
			<td>Use in resistance mode and maintain unplugged during TER measurements</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>MEARIENFELD</td>
			<td>610610</td>
			<td>Hemocytometer&#160;</td>
		</tr>
		<tr>
			<td>Leica TCS_SP5_MO</td>
			<td>Leica</td>
			<td>Without/number</td>
			<td>Laser confocal microscopy with Leica microsystems CMS Gmbh/leica Las af Lite/BIN software</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td>Mounting medium for fluorescence</td>
		</tr>
		<tr>
			<td>4&#180;, 6-diamino-2-phenylindole (Dapi)</td>
			<td>SIGMA</td>
			<td>D-9542</td>
			<td>0.05 mM final concentration</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A-1310</td>
			<td>0.5%&#160; final concentration for blocking solution</td>
		</tr>
	</tbody>
</table>

analysis of the epithelial damage produced by entamoeba histolytica infection

阿米巴是人类阿米巴病的病原体，腹泻和肝脓肿在热带国家的重要原因。感染是通过与肠上皮细胞的病原体的相互作用启动。这种相互作用导致细胞间的结构，例如紧密连接（TJ）的中断。 TJ保证上皮细胞层，以宿主组织从肠腔中分离的密封。最近的研究提供的证据表明由寄生蛋白EhCPADH112中断TJ的是大肠杆菌的先决条件阿米巴入侵是伴随着上皮屏障功能障碍。因此， 大肠杆菌中参与TJ拆卸分子机制的分析阿米巴入侵是非常重要的改善我们的阿米巴病发病机制的认识。本文介绍了一个简单的模型，它允许初始宿主 - 病原体相互作用的评估和寄生虫侵袭潜能。要分析的参数包括Transepithelial电阻，EhCPADH112与上皮细胞表面的受体，改变表达与上皮交界标记和上皮细胞内的寄生虫分子的本地化本地化互动。

对于一个成功的E。阿米巴文化，有两个重要条件必须满足:成长在无菌条件下与收获对数生长期。 E.以前，文化阿米巴是容易建立与细菌或锥虫22的某些物种的关联。但是，现在它通常有无菌培养这种寄生虫的意思变形虫在免费代谢细菌，真菌，原生动物，后生动物或细胞环境中的不确定传代培养。此外，后期对数生长早期稳定期收获滋养体关键是有活力和增殖细胞<sup...

Watch this Scientific Journal Video about Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection at JoVE.com

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

人感染阿米巴导致阿米巴病，腹泻在热带国家的重要原因。感染是通过与肠上皮细胞病原体相互作用启动，引起的细胞 - 细胞接触的开口，因而腹泻，有时接着肝感染。本文提供了一个模型来评估早期的宿主 - 病原体相互作用，以改善我们的阿米巴病发病机制的认识。

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

analysis-epithelial-damage-produced-entamoeba-histolytica

Research

JoVE Journal

Immunology and Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

18.6K 次观看.  Center for Research and Advanced Studies of the National Polytechnic Institute. 人感染阿米巴导致阿米巴病，腹泻在热带国家的重要原因。感染是通过与肠上皮细胞病原体相互作用启动，引起的细胞 - 细胞接触的开口，因而腹泻，有时接着肝感染。本文提供了一个模型来评估早期的宿主 - 病原体相互作用，以改善我们的阿米巴病发病机制的认识。 

视频: Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

半胱氨酸残基的溶剂可及性分析<em&gt;玉米rayado菲诺病毒</em病毒样颗粒<em&gt;烟草</em植物和交联的肽类病毒颗粒

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

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 ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

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 ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...