这项工作是由加拿大健康研究，通过催化剂的联合感染和并发症补助研究院支持。人红细胞提供了中心医院DE L&#39;拉瓦尔大学血库。

注：与HIV-1和恶性疟原虫的实验必须在适当的生物安全水平实验室（BSL2寄生虫，BSL3 HIV-1和共同感染），可能受感染的人体血液中使用时，必须采取特殊的预防措施。 1。外周血单个核细胞（PBMC）的分离纯化（基于8日和9） <ol><li>开始从新鲜的人体血液与抗凝剂（肝素，柠檬酸，柠檬酸盐葡萄糖或柠檬酸磷酸葡萄糖）治疗的健康献血者（500毫升）。净化和整个协议的其余部分隔离PBMC和使用无内毒素的试剂。 </li><li>用70％乙醇消毒，包括管，血袋，切管，小心地转移到T150大掌柜瓶血。 </li><li>准备15毫升淋巴细胞分离介质，每50毫升锥形管（确保Ficoll分离已经达到室温）（Ficoll分离）16管。仔细层顶部的新鲜血液30毫升。 </li><li>离心30分钟在400 XG在20°Ç折断。 </li><li>在离心，准备8×50 ml锥形管，室温汉克的平衡盐溶液（HBSS中）每管25毫升。 </li><li> 仔细接口多云细胞环（包含PBMC），转移至50毫升的试管中的HBSS（池2每管细胞环）。完成量上升至50 ml HBSS中。 </li><li>离心15分钟，在150 XG在20°Ç上休息。 </li><li>在阴天的细胞环离心，收集黄色上层Ficoll分离的步骤，血浆池，填补了50毫升锥形管。灭活血浆中补充50毫升管加热在56℃，30分钟旋转10分钟为1800 xĞ。保持上清（含稀释自体血浆可以作为介质在稍后的步骤的补充）。 </li><li> 小心取出上清液和resuspenð从步骤1.7在25毫升的HBSS和池2颗粒每50毫升管细胞沉淀。在8分钟的300 XG旋转20°C </li><li>小心去除上清，并在10毫升的HBSS和池在一个50毫升管的悬浮颗粒。 </li><li>采取等份，进行台盼蓝排斥，以评估死亡率和再算上PBMC中。 </li><li>完整的体积为50毫升的HBSS和自旋10分钟300×Ğ。去除上清，悬浮在5X10 6细胞/ ml的细胞在RPMI 1640（应该约400-500×10 6 PBMC中获得500毫升血液）。 </li></ol> 2。单核细胞源性巨噬细胞（MDM）的分化（基于8日和9） <ol><li>种子约1.25×10 8 PBMC的RPMI 1640培养液（25毫升/皿）在15厘米菜。 </li><li>让细胞坚持1-2小时，在37°C，5％CO 2气氛。 </li><li>转让非贴壁细胞，在一个新瓶，并用15毫升内毒素免费PBS两次（5板分钟，300 XG）。贴壁细胞是孤立的单核细胞。 </li><li>为了让新鲜分离的单核细胞分化成的MDM，加入20毫升5％自体血浆（从1.8步）的RPMI 1640培养基。加入重组人巨噬细胞集落刺激因子（M-CSF的25毫微克/毫升）和青霉素/链霉素溶液（聚苯乙烯，100 U / mL青霉素，100μg/ mL链霉素）。孵育2天，改变培养基孵育2-3额外天在37°C，5％的CO 2气氛。 </li><li>删除旧的介质，并用内毒素无PBS一次，添加的PBS轻轻立即吸。要分离的MDM充分分​​化，治疗在37°C〜7毫升Accutase（Accutase，蛋白酶和胶原溶解的活性，使细胞从塑料盘支队市售的混合）在5％CO2的细胞为15-20分钟2的气氛，在中旬时轻轻摇动。 </li><li>在每盘添加3-5毫升自体血浆（从1.8步）（以帮助保护细胞刮）。 </li><li>轻轻刮去确保介质覆盖在任何时候，在转让50毫升管/池细胞的细胞。 </li><li>用大量的内毒素无PBS板恢复尽可能多的细胞。 </li><li>旋转200 XG 5分钟，弃上清。 </li><li> 5毫升MDM培养基（10％灭活补胎牛血清白蛋白（iFBS），聚苯乙烯，25毫米肝素钠补充）和计数细胞（MDM的产生通常是2-8％总的PBMC）的RPMI 1640重悬沉淀。注：在这一步可能会采取的MDM等分，以评估流式细胞仪检测细胞纯度人口，通常获得99％的MDM的快递CD14的。 </li><li>种子1×10 5的MDM MDM的文化介质在24孔板，每孔600μL。 </li><li> 37℃孵育过夜在5％的CO 2感染前的气氛。 </li></ol> 3。生产和定量的HIV-1病毒的股票<ol><li>生产中使用磷酸钙转染HEK293T细胞follo病毒股翼福尔廷等协议10。 </li><li>为了获得20微克任NL4.3 吕克 + 环境保护与单周期内含有编码荧光素酶报告基因的病毒，共同转染HEK293T细胞- R +和的pHCMV-G的10微克，或15微克NL4.3 吕克环境保护 - R +和15微克pJRFL ENV。使用30微克的NL4.3 吕克 + 环境保护- R的+生成控制糖蛋白缺陷病毒。对于完全复制病毒，使用30微克的NL4.3Bal ENV。向量pHCMV-G的和pJRFL ENV编码水泡性口炎病毒包膜糖蛋白（VSV-G的）和1 CXCR5的嗜性HIV-1（比NL4.3Bal ENV不同），分别。可从8质粒的额外信息。质粒可通过美国国立卫生研究院艾滋病研究和参考程序（NIAID的，NIH）。 </li><li>所有使用病毒酶联免疫吸附股票量化的HIV-1 P24衣壳蛋白11，并在使用前确认其感染的潜力。我们评估我们储存的天花病毒的传染性，通过使用TZM-BL指标细胞12。 </li></ol> 4。文化疟原虫的寄生虫（13） 4.1寄生虫一般文化和维护<ol><li>保持体育恶性疟原虫 3D7人类红细胞1的RPMI 1640 4％血细胞比容辅以0.5％（W / V）Albumax二，25微克/毫升庆大霉素（25毫米肝素钠和25毫米碳酸氢钠3缓冲）（血组Ø+）的无性阶段的寄生虫370μM次黄嘌呤在37°C在1％氧气/氮气中5％的二氧化碳的混合气体。 </li><li>原虫可以监视由薄涂片的文化，有15％姬姆萨液染色。 </li><li>始终保持在未受感染的红血细胞在相同条件下（uRBC）培养皿寄生虫作为控制使用。 为了获得后期parasitES（滋养体/早期裂殖），同步的寄生虫如下： </li></ol> 4.2寄生虫同步<ol><li>在早晨，收获寄生虫文化，在300 XG旋转5分钟，弃上清。 </li><li> 5包装的细胞量在5％（W / V）的D-山梨醇，并培育5分钟在37°C重悬浮颗粒</li><li>旋转300×Ğ5分钟，丢弃30毫升的培养基上清，重悬沉淀，并在孵化器回。 </li><li>约8小时后，重复步骤4.2.1至4.2.3为了获得紧密同步的寄生虫。后期寄生虫（滋养体/早期裂殖），然后可以收获第二天早上进行的实验。净化前，进行姬姆萨染色，以确认生命周期阶段。 </li></ol> 4.3 恶性疟原虫感染的红血细胞（iRBC）净化<ol><li>消毒用70％乙醇和p VarioMacs分离器（支架和磁铁）花边生物罩下。 </li><li>修复三通阀CS列的底部。 </li><li>切针与美天旎的特殊刀具，塑料保护和修复针活塞底部。 </li><li>磁铁锁入列仔细。 </li><li> MDM培养基10毫升，用拧左边的活塞套件封闭注射器慢慢注入删除列中的所有气泡。 </li><li>用〜20毫升MDM培养基列。 </li><li>收获培养板中的红细胞，洗一次10毫升的MDM培养基（300 XG，5分钟）和红细胞颗粒悬浮在12毫升的MDM培养基。慢慢地添加到列中，并通过让悬浮流（只感染的红血细胞中含有寄生虫滋养体或裂殖阶段，将保留的磁场由于存在hemozoin晶体）。 </li><li>洗一次20毫升中，当它到达列顶部的白色圆盘，停止液体流动。 </li><li>删除列磁铁和12毫升MDM培养基在一个干净的无菌管流出iRBC。 </li><li>涂抹等份的恢复iRBC并进行姬姆萨染色，以确认生命周期阶段。 </li><li>计数恢复iRBC使用血球（隔离红细胞是100％iRBC）。 </li><li>颗粒细胞（300 XG，5分钟），弃上清和细胞治疗500μL新鲜AB +人血清（opsonisation第14步）。 </li><li>在37°C，5％的CO 2气氛中孵育30分钟。 </li><li>洗一次10毫升MDM的文化媒介（300× 克 ，5分钟），所需浓度重悬iRBC。通常，10％，原虫15厘米培养皿大致给出〜0.5 1×10 8紧密同步iRBC。 </li></ol> 5。暴露于疟原虫的MDM <ol><li>的MDM揭露到uRBC或iRBC的，细胞必须被悬浮在MDM培养基中，以获得一个uRBC / iRBC比例：75:1的MDM。在先前准备24孔板的MDM添加适当的红细胞悬浮液的体积，以每口井。执行一式三份的所有实验。 </li><li>在37°C，5％的CO 2气氛中孵育4小时的共同文化。 </li><li> 600μL内毒素的PBS 3次洗涤细胞，添加在PBS轻轻吸立即。 </li><li>裂解剩下的红细胞，加入200μl无菌水冰冷的20秒和吸洗井。 </li><li>加入600μLMDM培养基中培养24小时，在37°C，5％的CO 2气氛。 </li></ol> 6。完全复制型HIV-1感染的MDM <ol><li>为了评估体育的影响疟原虫在MDM，对HIV-1病毒复制增加，根据图1A，10毫微克的P24 NL4.3Bal 包膜病毒在适当的井（300μL的MDM媒体）所列的计划，只添加在控制井的MDM中等。 </li><li>在37°C孵育2小时5％CO 2气氛。 </li><li>洗细胞，内毒素的PBS 3次添加的PBS轻轻吸立即。加入600μl新鲜的MDM培养基每口井的。 </li><li>在37°C孵育5％的CO 2气氛中。 </li><li>在3天，6，9及12后，病毒感染的开始，收获每个上清200μL，加入200μl新鲜MDM介质的其后。保持在-20℃的收获上清，用ELISA福尔廷等10后的主要HIV-1衣壳p24蛋白定量。 </li></ol> 7。单周期病毒编码荧光素酶与感染的MDM <ol><li>以确定寄生虫的艾滋病毒在MDM-1基因表达的影响，添加，根据计划概述图1B，10纳克P24任NL4.3 吕克 + 环境保护 （300μL的MDM培养基） - R的+（ VSV-G的），NL4.3 luc +的环境保护- R的+（JRFL ENV）或NL4.3 吕克 +环境保护- R的+病毒在相应的井。 </li><li>在37°C孵育2小时，5％的CO 2气氛。 </li><li> 600μL内毒素的PBS 3次洗涤细胞，添加在PBS轻轻吸立即。加入600μl新鲜的MDM培养基每口井的。 </li><li>在37°C孵育5％的CO 2气氛中。 </li><li>删除中期的72小时以下的感染，并添加200μL裂解缓冲液1X（与荧光素酶检测试剂盒提供）。让细胞在室温下溶解，轻轻摇动。贮存于-20°C </li><li>解冻板和40微升裂解液转移到一个96光度计板的;添加荧光素酶底物100μL（与荧光素酶检测试剂盒提供）和测量的荧光素酶（萤火虫荧光素酶）在制造商的指示后光度计活动。 </li></ol> 8。代表结果用我们的共同感染模型，我们表明，exposurE的体育疟原虫到MDM的降低HIV-1感染的易感性。事实上，一个显着减少（P级&lt;0.05; 2路方差分析，12天）中释放病毒颗粒，HIV-1上清液中的P24衣壳蛋白衡量，观察预处理与寄生虫（ 图2A）在MDM。这一观察也证实由编码荧光素酶报告基因的病毒感染的细胞。 MDM的与窝藏无论是外源性的，VSV-G或HIV-1糖蛋白的这种病毒的感染导致显着（P &lt;0.05，学生t-检验）荧光素酶在细胞暴露在体育生产恶性疟原虫 （ 图2B）。值得注意的是，VSV-G假型病毒产生了更大的荧光素酶活性比他们JRFLenv同行，这是由于VSV-G的15假型粒子的感染效率。由于寄生虫博览会MDM的影响，这两种类型的病毒，这表明它影响一些病毒基因前一步pression（ 图2B）。同样重要的是提细胞活力没有影响的MDM博览会到iRBC（数据未列出），表明观察到的抑制是具体的，而不是由于细胞死亡率。 <img alt="图1" src="/files/ftp_upload/4166/4166fig1.jpg" /> 图1。一）为MDM感染板计划完全复制病毒的模拟：未受感染的细胞。 uRBC：细胞暴露给未受感染的红血细胞。 iRBC：细胞暴露到恶性疟原虫感染的红血细胞。 BAL：。NL4.3Bal ENV巴尔/ uRBC的感染细胞：细胞暴露到uRBC感染与NL4.3Bal ENV巴尔/ iRBC的： 暴露与ENV 乙 NL4.3Bal iRBC和感染的细胞）为MDM感染板计划单周期内的荧光素酶编码病毒的模拟：未受感染的细胞。 uRBC：细胞暴露给未受感染的红血细胞。 iRBC：细胞接触到疟原虫 -Infected红血细胞。 δenv：感染的细胞与NL4.3 吕克 + 环境保护- R的。JRFL：与NL4.3 吕克 + 环境保护感染的细胞- R的+（JRFL ENV）。 VSV-G：受感染的细胞与NL4.3 吕克 + 环境保护- R的+（VSV-G）Δenv/ uRBC：细胞暴露到uRBC和与感染NL4.3 吕克 + ENV-R +Δenv/ iRBC的细胞暴露iRBC感染NL4.3 吕克 + 环境保护- R的+ JRFL / uRBC：细胞暴露到uRBC和感染NL4.3 吕克 + 环境保护- R的+（JRFL ENV）。 JRFL / iRBC：细胞暴露到iRBC和感染NL4.3 吕克 + 环境保护- R的+（JRFL的ENV）。 vsv-g/uRBC：细胞暴露到uRBC和感染NL4.3 吕克 + 环境保护- R的+（VSV-G）vsv-g/iRBC：细胞暴露到iRBC和感染NL4.3 吕克 + 环境保护- R的+ （VSV-G）。 <img alt="图2" src="/files/ftp_upload/4166/4166fig2.jpg"/&gt; 图2。一）对恶性疟原虫对HIV-1号病毒在MDM生产的MDM暴露到uRBC或iRBC比率​​75:1（uRBC / iRBC：MDM）为4小时和广泛洗净。 ENV NL4.3Bal 2小时P24 10ng细胞感染病毒生产用ELISA监测HIV-1在不同的时间点，最初的病毒感染后无细胞上清的P24。一个有代表性的试验显示B）对恶性疟原虫对HIV-1号病毒的转录在MDM MDM的比率75:1（uRBC / iRBC：感染与uRBC或iRBC的。，MDM）。细胞，然后感染与10ng P24单周期病毒（或者NL4.3 luc +的环境保护- R的+，NL4.3 吕克 + 环境保护 - R +（JRFL ENV）或NL4.3 luc +的环境保护- R的+（VSV- g）条）。荧光素酶的表达进行了评估，在细胞裂解物72小时后，最初的病毒感染。一个有代表性的实验证明。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+hemozoin+modulates+susceptibility+of+immature+monocyte-derived+dendritic+cells+to+HIV-1+infection+by+inducing+a+mature-like+phenotype.">Malaria hemozoin modulates susceptibility of immature monocyte-derived dendritic cells to HIV-1 infection by inducing a mature-like phenotype.</a> Cell Microbiol. 12, 615-625 (2010).</li><li>Diou, J., Gauthier, S., Tardif, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+the+malaria+pigment+hemozoin+renders+human+macrophages+less+permissive+to+HIV-1+infection.">Ingestion of the malaria pigment hemozoin renders human macrophages less permissive to HIV-1 infection.</a> Virology. 395, 56-66 (2009).</li><li>Davies, J. Q., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+human+macrophages.">Isolation and culture of human macrophages.</a> Methods Mol. Biol. 290, 105-116 (2005).</li><li>Fortin, J. F., Cantin, R., Lamontagne, G., Tremblay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+ICAM-1+glycoproteins+incorporated+on+human+immunodeficiency+virus+type+1+are+biologically+active+and+enhance+viral+infectivity.">Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity.</a> J. Virol. 71, 3588-3596 (1997).</li><li>Bounou, S., Leclerc, J. E., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J. Virol. 76, 1004-1014 (2002).</li><li>Wei, X., Decker, J. M., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+resistant+human+immunodeficiency+virus+type+1+in+patients+receiving+fusion+inhibitor+(T-20)+monotherapy.">Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.</a> Antimicrob. Agents Chemother. 46, 1896-1905 (2002).</li><li>Trager, W., Jensen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+malaria+parasites+in+continuous+culture.">Human malaria parasites in continuous culture.</a> Science. 193, 673-675 (1976).</li><li>Turrini, F., Ginsburg, H., Bussolino, F., Pescarmona, G. P., Serra, M. V., Arese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+Plasmodium+falciparum-infected+human+red+blood+cells+by+human+monocytes:+involvement+of+immune+and+nonimmune+determinants+and+dependence+on+parasite+developmental+stage.">Phagocytosis of Plasmodium falciparum-infected human red blood cells by human monocytes: involvement of immune and nonimmune determinants and dependence on parasite developmental stage.</a> Blood. 80, 801-808 (1992).</li><li>Akkina, R. K., Walton, R. M., Chen, M. L., Li, Q. X., Planelles, V., Chen, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+CD34++cells+with+a+human+immunodeficiency+virus+type+1-based+retroviral+vector+pseudotyped+with+vesicular+stomatitis+virus+envelope+glycoprotein+G.">High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.</a> J. Virol. 70, 2581-2585 (1996).</li><li>Zhao, C., Papadopoulou, B., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+infantum+enhances+human+immunodeficiency+virus+type-1+replication+in+primary+human+macrophages+through+a+complex+cytokine+network.">Leishmania infantum enhances human immunodeficiency virus type-1 replication in primary human macrophages through a complex cytokine network.</a> Clinical Immunol. 113, 81-88 (2004).</li></ol>

没有利益冲突的声明。

我们这里介绍两种不同的方法来分析，通过分析或者病毒基因的表达或子代病毒的生产和单核细胞源性巨噬细胞中的复制的HIV-1病毒的周期，即疟疾寄生虫的影响。类似的方法已被用于其他的HIV-1寄生虫合并感染16。然而，这些新的数据是在疟疾HIV-1感染的调查一步。 。事实上，迪欧等 8研究的hemozoin的效果，对HIV-1病毒复制不活的寄生虫，在与我们的研究结果一致，他们观察到，hemozoin本身并不足以抑制病毒的MDM生产，而不是MDMS。 使用描述实验的布局，我们观察到， 体育恶性疟原虫对明确的巨噬细胞上的HIV-1复制周期的不利影响：预先接触到寄生虫的巨噬细胞中观察到的病毒生产的显着抑制（Figuré2A）和寄生虫病毒转录的具体影响在图2B所示。但是，我们不能离开了病毒进入或融合（decapsidation），或整合后的机制，如蛋白质的合成或病毒粒子组装和出芽，寄生虫任何额外的效果。此外，它是可能的，如果在同一时间或以下的MDM病毒感染寄生虫，将获得对不同HIV-1复制的影响。 我们提供了一个强大的工具进行详细调查的HIV-1 / P 在体外共感染模型疟原虫在宿主细胞相互作用。例如，这与其他技术，如实时定量PCR技术，它可以目标和量化在的病毒retrotranscription和病毒基因整合到宿主细胞基因组的具体步骤，实验布局相结合，是相当可行的，并应取得进一步insights将参与共同感染的机制。此外，具体分析，以评估病毒的周期（病毒融合，decapsidation;，进入定量）的早期步骤，可以适用于这一基本协议，以进一步分析病毒复制的效果。这些修改表明，该协议是关于如何灵活HIV-1的定量分析和检测：确实，即使是标准的HIV-1逆转录酶定量检测用氚标记的核苷酸，可以想象取代ELISA检测为HIV-1 P24。除了MDM的，我们希望我们的系统能适应HIV-1疟疾，如单核细胞和树突状细胞相互作用有关的其他类型的细胞。事实上，MDM的贴壁细胞，促进他们的操作，容易使洗涤iRBC尚未采取，或在接触与MDM，不排除任何的巨噬细胞。这种优势并不适用于树突状细胞和单核细胞，悬​​浮培养，复杂的SEparation的uRBC和免费用这样的细胞，我们目前正在解决这个问题iRBC。我们的系统也可能是有用的研究更复杂的相互作用，如疟原虫暴露在其他免疫细胞CD4阳性T细胞共培养实验，如HIV-1病毒的循环单核细胞源性细胞的影响。最后，我们的协议可能是适合看体育的影响实验HIV-1感染者的HIV-1活化PBMC中的恶性 iRBCs为。 伦理声明人PBMC获得了从健康献血者的血液，在按照中心医院DE L&#39;拉瓦尔大学研究中心的生物伦理委员会的指导方针。从所有献血者获得书面同意。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> RPMI 1640培养</td><td>多节</td><td> 350-000-CL </td><td></td></tr><tr><td> PBS-内毒素</td><td>西格玛</td><td> D8662 </td><td></td></tr><tr><td>胎牛血清</td><td>欧洲野牛</td><td> 080-150 </td><td>热灭活56°C时为30分钟</td></tr><tr><td> accutase </td><td> eBiosciences </td><td> 00-4555-56 </td><td></td></tr><tr><td> albumax二</td><td> Gibco公司</td><td> 11021-037 </td><td>在RPMI 1640溶解，过滤，消毒</td></tr><tr><td>淋巴细胞分离液</td><td>多节</td><td> 305-010-CL </td><td></td></tr><tr><td>白色光度计板</td><td> Promega公司</td><td> Z3291 </td><td></td></tr><tr><td>政务司司长的Mac分离columms </td><td>美天旎生物技术</td><td> 130-041-305 </td><td></td></tr><tr><td> VarioMacs分隔</td><td>美天旎生物技术</td><td> 130-090-282 </td><td></td></tr><tr><td>青霉素/链霉素溶液</td><td>欧洲野牛</td><td> 450-201-EL </td><td></td></tr><tr><td> D-山梨醇</td><td> Sigma-Aldrich公司</td><td> S1876 </td><td></td></tr><tr><td>细胞刮刀（25厘米） </td><td>萨尔斯塔特</td><td> 83.1830 </td><td></td></tr><tr><td> 24孔细胞培养板</td><td>屋宇署猎鹰</td><td> 353047 </td><td></td></tr><tr><td> 150×20毫米培养皿</td><td>萨尔斯塔特</td><td> 83.1803.003 </td><td></td></tr><tr><td> M-CSF的</td><td>金斯瑞</td><td> Z02001 </td><td></td></tr><tr><td>肝素钠</td><td> Sigma-Aldrich公司</td><td> H4034 </td><td></td></tr><tr><td>人血清AB + </td><td>谷生物医学</td><td> HP1022 </td><td></td></tr><tr><td> HBSS中</td><td>欧洲野牛</td><td> 311-505-CL </td><td></td></tr><tr><td>庆大霉素</td><td> Sigma-Aldrich公司</td><td> G1397 </td><td></td></tr><tr><td>次黄嘌呤</td><td> Sigma-Aldrich公司</td><td> H9636 </td><td></td></tr><tr><td> 碳酸氢钠 </td><td> Sigma-Aldrich公司</td><td> S5761 </td><td></td></tr><tr><td>萤光素酶检测系统</td><td> Promega公司</td><td> E1500 </td><td></td></tr><tr><td>人体红血细胞</td><td></td><td></td><td>获得纯化哲血库。 </td></tr></tbody></table>

an in vitro co-infection model to study plasmodium falciparum-hiv-1 interactions in human primary monocyte-derived immune cells

致命的形式，疟疾的病原体疟原虫 ，人类免疫缺陷病毒类型1（HIV-1）是全球最重要的健康问题，负责一共有4万人死亡，每年1。由于其广泛的重叠，在发展中地区，特别是撒哈拉以南非洲，疟疾和HIV-1病毒的感染是常见的，但是这两种疾病之间的相互作用是了解甚少。流行病学报告表明，疟疾感染瞬时增强HIV-1复制和增加HIV-1病毒共同感染者的负荷2,3。因为这种病毒血症保持高抗疟药物治疗后的几个星期，这种现象可能有疾病的进展和传输的影响。 这些意见背后的细胞免疫机制的研究已经只有很少。 在体外研究investig包括生成疟疾，HIV-1的影响已经表明，接触可溶性疟疾抗原可以增加HIV-1感染和免疫细胞的激活。然而，这些研究使用全细胞提取物， 体育恶性疟原虫裂殖阶段寄生虫和外周血单个核细胞（PBMC）中，难以破译，疟疾组件（S）是负责观察到的效果和目标主机的细胞4,5。最近的工作表明，不成熟的单核细胞来源的树突状细胞的疟原虫色素hemozoin曝光，增加了他们的能力转移HIV-1的CD4 + T细胞6,7，但它降低HIV-1感染的巨噬细胞8。为了阐明这个复杂的过程，疟疾寄生虫和HIV-1在不同的有关人权的主要细胞群之间的相互作用的系统的分析，是迫切需要的。 调查的H影响的几个技巧IV-1对微生物的吞噬功能，这些病原体对HIV-1复制的影响已被描述。在这里，我们提出了一种方法，探讨体育的影响恶性疟原虫感染的红细胞，在人类原发性单核细胞源性巨噬细胞中HIV-1病毒的复制。监测使用单周期在荧光素酶报告基因取代env基因的假型病毒，而病毒感染的巨噬细胞释放的数量上的影响是通过测量确定寄生虫暴露在HIV-1的转录/翻译活动的影响HIV-1衣壳蛋白P24在细胞培养上清用ELISA法。

Watch this Scientific Journal Video about An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells at JoVE.com

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

我们已经制定了一个<em&gt;在体外</em&gt;疟疾，HIV-1病毒共感染模型研究的影响<em&gt;恶性疟原虫</em&gt;在人类原发性单核细胞源性巨噬细胞的HIV-1病毒复制周期。这种多用途的系统可以很容易地适应其他主要易感HIV-1感染的细胞类型。

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

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Research

JoVE Journal

Immunology and Infection

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

13.6K 次观看.  CHUL (CHUQ), Quebec City, Quebec, Canada. 我们已经制定了一个在体外疟疾，HIV-1病毒共感染模型研究的影响恶性疟原虫在人类原发性单核细胞源性巨噬细胞的HIV-1病毒复制周期。这种多用途的系统可以很容易地适应其他主要易感HIV-1感染的细胞类型。

视频: An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.