我们要感谢国家科学基金会拨款（CBET-0846259）。

请参阅表1，准备在这个协议中引用的所有媒体，缓冲区和解决方案。 1。生产的AHA-标记的腺病毒<ol><li>准备人类胚胎肾脏细胞11 100毫米组织培养皿（HEK 293）（ 见表1）在HEK 293细胞的生长介质保持在37°C，直到他们达到80 - 90％汇合。 （注:文化的汇合90％以上的感染，可能不会产生所需的病毒可能很难。） </li><li>松开的菜肴之一，首先消除的生长介质，5毫升的TD缓冲区冲洗一次，然后孵化一分钟1毫升的胰蛋白酶-EDTA（1×）的细胞的细胞。 </li><li>加入9毫升的HEK 293细胞的生长介质，并使用血球计数仪，指望从这个菜收获HEK 293细胞的数量。使用这个数字来计算金额感染腺病毒5型（Ad5的），使用1 multiplicit感染Y（MOI），价值10 PFU /细胞。账户的事实，Ad5的正常，传染性指数（传染性微粒的粒子总数的比例）通常是1:20。 </li><li>准备感染缓冲区10毫升。 </li><li>慢慢地从其余十培养皿HEK293细胞生长培养基，然后用5毫升的TD缓冲区。加入1毫升感染的缓冲区，以每道菜在37°C孵育1小时。在此期间感染期间，轻轻摇动每个培养皿一边到另一边，每隔15分钟。 </li><li>感染后在37°C，加入9毫升新鲜的HEK 293细胞生长培养基孵育18小时。 </li><li>编制阿哈标签介质（或符合控制介质控制）100毫升。务必过滤0.2微米的无菌注射器过滤器前准备的标签媒体集中的股票解决方案。 </li><li>仔细（以免洗去感染的细胞），取出后18 HR的HEK293细胞生长培养基第二洗每个培养皿中，用5毫升的TD缓冲区。 </li><li>删除TD缓冲洗涤液，每个培养皿中加入10毫升的阿哈标签介质。蛋氨酸控制介质控制，应在这一步，而不是的阿哈标签介质。 </li><li>标签介质在37°C为6小时的孵育感染的细胞。 ，随时标签可以在12 - 24小时后感染，但不应该是持续时间超过6小时的时间。此时间间隔已经确定，最大限度地与病毒结构蛋白翻译重叠，确保传染性不显着减少。7 </li><li>小心地取下标签介质，以免破坏细胞。新鲜HEK293细胞生长培养基中加入10毫升，每道菜，另外18小时在37°C孵育细胞。受感染的细胞在这一步，以尽量减少损失，这是完全没有必要删除标签解决方案。 HEK293细胞的培养基中含有过量的M等将outcompete任何剩余阿哈。 </li><li>继续加强净化3。 </li></ol> 2。生产的叠氮糖标记的腺病毒<ol><li>按照题为阿哈标记的腺病毒，包括步骤1.5生产过程。 </li><li>准备100毫升的叠氮糖标签介质。 </li><li>覆盖在37°C细胞10毫升新鲜叠氮糖标签介质和孵育44小时。 </li></ol> 3。叠氮化物标记的腺病毒颗粒的分离纯化<ol><li>从每个培养皿和锥形管转移到受感染的细胞取出媒体。在2000克离心10分钟，在4°C。取出上清液和悬浮颗粒在TD缓冲区（使用TD每10道菜的缓冲区8毫升）。 </li><li>重复（3个或更多）冻融循环使用杜瓦瓶液氮和37℃水浴裂解感染细胞。在离心2000克10分钟在4°C，从上清中分离出来的细胞碎片。 </li><li>准备先成超清晰的离心管中吸取2.5毫升1.25克/毫升的氯化铯溶液1 CsCl密度梯度离心管。接下来，使用其尖端定位在试管底部吸管慢慢加入2.0毫升1.4克/毫升的氯化铯溶液，小心不要打扰梯度界面。 </li><li> CsCl密度梯度管的顶部3.2从步骤上，吸取上清液，如果有必要附加的TD缓冲区，以填补管。摆动斗在125000克的转子为1小时15°在超高速离心Ç离心样品。 </li><li>超速完成后，应该是在氯化铯的两个解决方案的界面作为一个厚厚的白色条纹可见病毒颗粒。做一个解决漏滴用无菌针管底部的小穿孔。 </li><li>收集了厚厚的白色带，其中应包含标记Ad5的颗粒，羚牛Ğ照顾，以排除多余的氯化铯的解决方案。避免收集细胞蛋白，这将带病毒层以上。此外排除空病毒粒子衣壳形成了较轻的一层厚厚的白色病毒带略高于。 </li><li>另外，执行对Ad5的粒子标记的最终净化。加入收集的分数从3.6步超离心管4毫升和1.35克/毫升的氯化铯溶液的填充管。离心125000克为18小时，于15°C，并收集在中间的步骤3.5和3.6所述的管形成厚厚的白色带。 （注意:如果病毒滴度低，一条白色带可能不可见第二离心后，将不可能恢复标记的病毒）。 </li><li>对于存放过长时间的病毒粒子，所收集的病毒提取透析应进行对病毒的存储缓冲区。透析的病毒样本储存在-80°C </li></ol> 4。里使用应变促进叠氮炔环加成炔探头修饰的腺病毒颗粒gation（SPAAC） <ol><li>到50μL病毒的存储缓冲区的Ad5的改性叠的解决方案，增加0.25μlSPAAC标签解决方案的。 （请参阅市售紧张炔商贩名单试剂表）。 </li><li>允许应变推动的环加成反应，继续在室温下过夜。如果使用光敏感探头（ 如 :TAMRA），包括反应容器用铝箔。 </li><li>净化标记的病毒，根据步骤6。 </li></ol> 5。结扎，炔探头在无氧大气中使用的铜（I）催化叠氮，炔环加成（CuAAC）修饰的腺病毒颗粒<ol><li>地方7.2毫克的铜（I）溴化物（溴化亚铜）Eppendorf管中的粉末，用铝箔覆盖。 </li><li>吸取1毫升DMSO成为第二个Eppendorf管。 </li><li>准备标签的CuAAC溶液50μl的在第三个Eppendorf管中。用铝箔覆盖，如果炔探头是光敏感（ 如 :TAMRA-炔）。 </li><li> 6小时3个Eppendorf管，不封顶，将在无氧手套袋。所有样品应保持里面的手套袋，直到完成的CuAAC反应。 </li><li>准备加入1毫升的DMSO 7.2毫克溴化亚铜粉末，这两个已经按照步骤5.4无氧的溴化亚铜50×原液。 </li><li>启动此溴化亚铜原液加入1μl移液器50μL的CuAAC标签解决方案，并已按照步骤5.4无氧CuAAC反应。使反应进行12小时内手套袋。 </li><li>净化标记的病毒，根据步骤6。 </li></ol> 6。标记病毒颗粒的纯化和贮存<ol><li>净化标记的病毒样本，使用离心九月凝胶过滤自旋Çolumns平衡与病毒存储缓冲区，按照制造商的指引。 </li><li>另外，可以进行净化透析对病毒的存储缓冲区。 </li><li>应储存在-80°C标记的病毒颗粒</li></ol> 7。标记的腺病毒荧光凝胶扫描的SDS-PAGE <ol><li>纯化标记病毒，煮沸10分钟至20μL加入20μLLaemmli样品缓冲液（2×）。样品离心30秒，以消除任何不溶性的沉淀。如果残余铜是目前净化后，沸腾的样品可以导致SDS-PAGE凝胶上蛋白的粗条纹，导致在可视化的困难。在这种情况下，放弃在此步骤中样品的沸点。 </li><li>将凝胶电泳细胞的录像带，并添加运行缓冲。病毒的蛋白质样品，装入井所需的数量。使用一个加载前STAINED分子量标记。运行1小时凝胶在200五，用铝箔覆盖整个期间保持电泳细胞减少的漂白的荧光标签。 </li><li>为最佳分辨率，并删除所有无荧光，允许运行跟踪染料的凝胶电泳。停止电泳和快速传输凝胶荧光凝胶扫描仪扫描凝胶在适当波长（574纳米）为:TAMRA荧光发射。 </li><li>每个病毒颗粒结合的染料分子的数量可以通过测量荧光的几个标准:TAMRA浓度和样品比较，从步骤7.3量化。 </li></ol> 8。代表结果 SDS-PAGE电泳和荧光凝胶扫描共轭:TAMRA-Ad5的观测结果如图3所示。虽然这些荧光的凝胶扫描进行样品作案通过CuAAC田间，结果应该是比较时探头通过SPAAC结扎。这些凝胶扫描表明，在阿哈三个Ad5的衣壳蛋白（Hexon蛋白，聚氯，纤维）的修改标签的结果，而糖的标签修改只有光纤衣壳蛋白。此外，增加在阿哈浓度的（4毫米与32毫米）的结果更大程度在阿哈成立的，但它也已显示，以减少传染性。前7结果表明，在4mm生产Ad5的阿哈，大约有5％的暴露蛋氨酸网站的是:TAMRA修饰，这个数字提高到10％时使用32毫米阿哈是。这相当于约280和510，分别为每Ad5的粒子，染料标记网站。糖标签1的分析表明，大约22 36 Ad5的糖基化位点是由叠氮糖占领，随后:TAMRA标记。 如果10个组织培养皿用于生产叠改性Ad5的描述<html>在这个协议中，200 - 300μL的叠氮化改性病毒应获得3.6步骤中收集的白色带，以滴度约为1.5×10 12颗粒/毫升。此过程的一个共同困难是缺乏病毒带在此纯化步骤，这可以归因于病毒滴度低。这是一般是过高或过低合流，或洗去细胞的感染细胞的结果 - 成为从感染后板松动而在第1步 - 冲洗。 <img src="/files/ftp_upload/4246/4246fig1.jpg" alt="图1" /> 图1。协议概述。叠氮含AHA或GalNAz首次被纳入到腺病毒颗粒。结扎与炔的荧光探针，然后通过"点击"化学。最后，SDS-PAGE电泳用于证明结扎荧光探针。 igure 2"/&gt; 图2。炔探头结扎上叠（一）SPAAC，（二）CuAAC下，含氧及（c）含氧气氛中通过修改病毒。 <img src="/files/ftp_upload/4246/4246fig3.jpg" alt="图3" /> 图3。荧光标记叠，腺病毒5型后CuAAC:TAMRA-炔共轭（Ad5的）的SDS-PAGE扫描。 （一）AHA-Ad5的标记，使用两种浓度阿哈。标签似乎发生腺病毒Hexon，聚氯和光纤衣壳蛋白，与阿哈浓度增加增加标签的程度。 （二）AC 4 GalNAz-标记的腺病毒似乎只能在光纤衣壳蛋白标记。 <table border="1"><tbody><tr><td> 解 </td><td> 准备 </td><td> 笔记 </td></tr><tr><td> AD5存储缓冲区</td><td> 5毫米的Tris-HCl缓冲液（pH 8.0）中含有0.5毫米50 mM氯化钠， 氯化镁 ，25％（V / V）甘油，0.05％（W / V）牛血清白蛋白（BSA）的</td><td>储存在-20°C </td></tr><tr><td> AD5感染缓冲区</td><td> 2％（V / V）小牛血清，1×TC的解决方案，在存储缓冲区AD5（量确定使用MOI 10 PFU /细胞和传染性指数1:20）。使用TD缓冲区最终体积带来的解决方案</td><td> Ad5的浓度可通过紫外吸收光谱测定。 </td></tr><tr><td> TC方案（200×） </td><td> 136毫米100毫米氯化钙 ，氯化镁2 </td><td>保存于4°C间</td></tr><tr><td>运输署缓冲区</td><td> 137 mM氯化钠，氯化钾5毫米，0.07毫米的Na 2 HPO 4，和25毫米的Tris-HCl，pH值7.5 </td><td>保存于4°C间</td></tr><tr><td> HEK293细胞生长培养基</td><td>辅以10％（V / V），小牛血清（BCS）的1％（V / V）笔链球菌杜尔贝科的改良Eagle培养基（DMEM培养基） </td><td>保存于4°C间</td></tr><tr><td>半胱氨酸原液（100×） </td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以200毫米L-半胱氨酸</td><td>准备新鲜和过滤0.2微米的无菌注射器过滤器在使用前。 </td></tr><tr><td>阿哈原液（25×） </td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以100毫米L-Azidohomoalanine </td><td>准备新鲜和过滤0.2微米的无菌注射器过滤器在使用前。 </td></tr><tr><td>阿哈标签介质</td><td> DMEM培养基（-蛋氨酸/半胱氨酸），辅以10％BCS，阿哈储备溶液（1×），2毫米L-半胱氨酸</td><td>使用前准备好新鲜。 这相当于一个4毫米Concentration啊哈，虽然不同浓度可用于（见代表结果 ）。 </td></tr><tr><td>气象原液（25×） </td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以100毫米L-蛋氨酸</td><td>准备新鲜和过滤0.2微米的无菌注射器过滤器在使用前。 </td></tr><tr><td>气象控制介质</td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以10％BCS的，符合储备溶液（1×），2毫米L-半胱氨酸</td><td>使用前准备好新鲜。 </td></tr><tr><td> AC 4 GalNAz，储备溶液（1000×） </td><td> 50毫米peracetylated的N - azidoacetylgalactosamine（AC 4 GalNAz）甲醇</td><td>保存于4°C。 AC 4 GalNAz是一个新陈代谢的前兆GlcNAz。 </td></tr><tr><td>叠氮糖标签介质</td><td> 100μLAC 4 GalNAz股票的解决方案，100毫升的HEK 293细胞生长培养基</td><td>允许甲醇蒸发之前加入HEK293细胞生长培养基。 </td></tr><tr><td>氯化铯解决方案</td><td>氯化铯溶解在TD缓冲区: 1.25克/毫升（18.08克/ 50毫升H 2 O） 1.35克/毫升（25.60克/ 50毫升H 2 O） 1.40克/毫升（31.00克/ 50毫升H 2 O） </td><td>密度梯度超速</td></tr><tr><td>病毒存储缓冲区</td><td>磷酸盐缓冲液（PBS），pH值7.2，含0.5毫米，0.9毫米氯化镁 ， 氯化钙 ，和10％（V / V）甘油</td><td>储存在-20°C </td></tr><tr><td> :TAMRA-BCN（SPAAC）的标签解决方案</td><td>在DMSO:TAMRA-BCN的: <ol><li>使用紫外线ABsorbance（外径260法）确定在步骤4.1滴度的病毒解决方案</li><li>在50μL等份确定的总数量（不适用Ad5的病毒颗粒） </li><li> :TAMRA-BCN的摩尔浓度（男）决定使用: çBCN的Ad5的 = 4×10 8×（不适用 /否AVO） 列印的AVO = 6.022×10 23 （在第4步准备，使反应混合物中包含100个病毒颗粒炔探头） </li></ol></td><td>应变推动炔探头的 对于SDS-PAGE电泳，约10 12颗粒/毫升的病毒滴度建议</td></tr><tr><td> :TAMRA-炔（CuAAC）的标签解决方案</td><td> 50μL叠改性Ad5的病毒存储缓冲区（Ad5的例确定为以上），1.5毫米batho<html>菲罗啉磺酸钠（ 即 1.5×最终溴化亚铜浓度）:TAMRA-炔。 :TAMRA-炔烃的摩尔浓度（米）的决定: çALK = 2×10 6×（N 重组腺病毒Ad5 / AVO） （在第5步准备，使反应混合物中包含100个病毒颗粒炔探头） </td><td> Tris缓冲被称为是一个有竞争力的铜和抑制配体8。 </td></tr><tr><td> Laemmli样品缓冲液（2×） </td><td> 125毫米的Tris-HCl（pH值6.8），4％（W / V）SDS，20％（V / V）甘油，10％（V / V）2 - 巯基乙醇，0.004％（W / V）溴酚蓝</td><td></td></tr><tr><td>运行缓冲液</td><td> 187毫米甘氨酸，3.5毫米，19毫米的Tris-HCl SDS H 2 O </td><td>保存于4°C间</td></tr></tbody></table>T" 表1。制备的缓冲区和本议定书所规定的解决方案。

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S., Lindholm, L., Boulanger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+Type+5+Fiber+Knob+Domain+has+a+Critical+Role+in+Fiber+Protein+Synthesis+and+Encapsidation.">Adenovirus Type 5 Fiber Knob Domain has a Critical Role in Fiber Protein Synthesis and Encapsidation.</a> J. Gen. Virol. 87, 3151-3160 (2006).</li><li>Zhang, M. M., Tsou, L. K., Charron, G., Raghavan, A. S., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+fluorescence+imaging+of+dynamic+S-acylation+and+protein+turnover.">Tandem fluorescence imaging of dynamic S-acylation and protein turnover.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 8627-8632 (2010).</li><li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, 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=Unnatural+amino+acid+incorporation+onto+adenoviral+(Ad)+coat+proteins+facilitates+chemoselective+modification+and+retargeting+of+Ad+type+5+vectors.">Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors.</a> J. Virol. 85, 7546-7554 (2011).</li><li>Hong, V., Presolski, S. I., Ma, C., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+Optimization+of+Copper-Catalyzed+Azide-Alkyne+Cycloaddition+for+Bioconjugation.">Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation.</a> Angew. Chem. Int. Ed. 48, 9879-9883 (2009).</li><li>Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry+and+Bioorthogonal+Reactions:+Unprecedented+Selectivity+in+the+Labeling+of+Biological+Molecules.">Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules.</a> Biochemistry. 48, 6571-6584 (2009).</li><li>Agard, N. J., Prescher, J. A., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Strain-Promoted+[3+++2]+Azide-Alkyne+Cycloaddition+for+Covalent+Modification+of+Biomolecules+in+Living+Systems.">A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems.</a> J. Am. Chem. Soc. 46, 15046-15047 (2004).</li><li>Dommerholt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Readily+Accessible+Bicyclononynes+for+Bioorthogonal+Labeling+and+Three-Dimensional+Imaging+of+Living+Cells.">Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells.</a> Angew. Chem. Int. Ed. 49, 9422-9425 (2010).</li><li>Link, A. J., Vink, M. K. S., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+the+functionalizable+methionine+surrogate+azidohomoalanine+via+copper-catalyzed+diazotransfer.">Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazotransfer.</a> Nat. Protoc. 2, 1879-1883 (2007).</li></ol>

没有利益冲突的声明。

在化学选择性和bioorthogonal点击反应，包括叠氮化合物的的发展是一个发展迅速的研究领域，以及随后出现的这些反应越来越多的选择，从生物耦合应用。我们已经限制了该协议的范围包括在我们自己的实验室和所有试剂的商业可用性的方法，因为它们的用途选择只有两个。 在第一个这样的路线-应变促进叠-炔环加成（SPAAC） -炔载内cyclooctyne环（ 图2a）。这是最近开发的方法，可以在含氧气氛下进行，并没有需要铜催化剂。9,10直到最近，这种方法的缺点之一是这些紧张炔费力的合成和减少商业可用性与末端炔铜（I）催化叠氮，炔环加成（铜AAC）;然而，越来越多的这些探头库是目前市售（见试剂表）。由于这些原因，我们选择了SPAAC在本协议作为首选方法。 还介绍了这个协议是结扎叠修改通过无氧作为螯合剂（ 图2b）使用bathophenanthroline二磺酸二钠盐（BDA）的气氛在CuAAC腺病毒颗粒。这种方法是在BDA的催化剂，是市售廉价方便。此外，报告的收益率一般最高可用的方法，产生活性氧的溶液中（参见含氧CuAAC，低于）所产生的并发症是可以避免的。然而，铜催化剂（一）被称为是细胞毒性， 11和需要专门的设备来实现这个过程可能会阻止一些团体使用。然而，我们发现这种方法的有效和有益的腺病毒修改。 第三CuAAC有用的方法最近已制定下工作的含氧大气条件（ 图2c）8。这种方法享有相同的福利作为BDA的螯合的方法，但作为螯合剂使用，以适应不大气中的氧气氧化破坏，而不是THPTA基板上。这种方法的一个小缺点是商业可用性的THPTA目前缺乏的，虽然简单，这种螯合剂合成已报告8。虽然我们没有我们自己的系统上执行此结扎法，THPTA / CuAAC预计将产生媲美北京经济技术开发区/ CuAAC，添加含氧大气宽容的便利。 尽管故意周围市售材料开发的这个协议，我们仍然要承认的成本优势和增加合成的灵活性此处使用叠氮化物和炔探头。 azidohomoalanine可以准备在低至3天，并显着高于商业来源。6,12紧张炔筹备工作更加困难11，但提供更大的探头选择的自由。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>腺病毒5型（Ad5的）含有绿色荧光蛋白转基因</td><td> BCBC </td><td> 391 </td></tr><tr><td>人类胚胎肾细​​胞（HEK 293） </td><td> ATCC </td><td> CRL-1573 </td></tr><tr><td>杜尔贝科的改良Eagle培养基（DMEM培养基），高糖</td><td> Invitrogen公司</td><td> 11965-092 </td></tr><tr><td>杜尔贝科的改良Eagle培养基，无蛋氨酸，没有半胱氨酸（DMEM-蛋氨酸/半胱氨酸），高糖</td><td> Invitrogen公司</td><td> 21013-024 </td></tr><tr><td>小牛血清</td><td> Invitrogen公司</td><td> 16170-078 </td></tr><tr><td>青霉素 - 链霉素100×解决方案（笔链球菌） </td><td> Invitrogen公司</td><td> 15140-122 </td></tr><tr><td> 0.5％胰蛋白酶-EDTA（10×） </td><td&gt; Invitrogen公司</td><td> 15400-054 </td></tr><tr><td> 100 MM细胞培养皿中，组织培养处理的聚苯乙烯</td><td>屋宇署猎鹰</td><td> 353003 </td></tr><tr><td>细胞培养二氧化碳培养箱</td><td></td><td></td></tr><tr><td>血球</td><td></td><td></td></tr><tr><td>生物安全柜</td><td></td><td></td></tr><tr><td>无菌注射器过滤器（0.2微米，醋酸纤维素） </td><td>厂商VWR </td><td> 28145-477（NA）514-0061（欧洲） </td></tr><tr><td>阿凡提乙脑离心机</td><td>贝克曼</td><td> 369001 </td></tr><tr><td>锥形管（50毫升，无菌） </td><td>屋宇署猎鹰</td><td> 352098 </td></tr><tr><td>管，薄壁，超清晰，13.2毫升，14 x 89毫米</td><td>贝克曼</td><td> 344059 </td></tr><tr><td>超速离心机配备与西南41和SW 60 Rotor </td><td>贝克曼</td><td></td></tr><tr><td> Eppendorf公司Biopur安全锁管，1.5毫升</td><td> Eppendorf公司</td><td> 0030 121.589 </td></tr><tr><td>离心机5418 </td><td> Eppendorf公司</td><td> 5418 </td></tr><tr><td>离心九月凝胶过滤离心柱</td><td>普林斯顿大学分离</td><td> CS-901 </td></tr><tr><td>无菌注射针，18针</td><td></td><td></td></tr><tr><td>氮手套袋（如果进行无氧CuAAC） </td><td></td><td></td></tr><tr><td>杜瓦瓶</td><td></td><td></td></tr><tr><td>液氮</td><td></td><td></td></tr><tr><td>电泳槽</td><td></td><td></td></tr><tr><td>荧光凝胶扫描仪</td><td></td><td></td></tr><tr><td>准备凝胶的Tris-HCl凝胶</td><td> BIO-RAD </td><td> 161-1105 </td></tr><tr><td> L - Azidohomoalanine </td><td> AnaSpec </td><td> 63669 </td></tr><tr><td>耶拿生物科学</td><td> CLK-AA005 </td><td></td></tr><tr><td> peracetylated 的 N-azidoacetylgalactosamine（AC 4 GalNAz） </td><td> Invitrogen公司</td><td> C33365 </td></tr><tr><td> Thermo Scientific的</td><td> 88905 </td><td></td></tr><tr><td> Sigma-Aldrich公司</td><td> A7480 </td><td></td></tr><tr><td> bathophenanthroline二磺酸（BDA）的钠盐</td><td> MP的Biomedicals </td><td> 0215011201 </td></tr><tr><td>二甲基亚砜（DMSO） </td><td></td><td></td></tr><tr><td>甲醇</td><td></td><td></td></tr><tr><td> Tris碱（三（羟甲基）氨基甲烷） </td><td></td><td></td></tr><tr><td>磷酸氢二钠（钠2 HPO 4） </td><td></td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td></td><td></td></tr><tr><td> 1M盐酸</td><td></td><td></td></tr><tr><td>甘油</td><td></td><td></td></tr><tr><td>牛血清白蛋白</td><td></td><td></td></tr><tr><td> L-半胱氨酸</td><td></td><td></td></tr><tr><td> L-蛋氨酸</td><td></td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td></td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td></td><td></td></tr><tr><td>甘氨酸</td><td></td><td></td></tr><tr><td>溴酚蓝</td><td></td><td></td></tr><tr><td>氯化铯（CsCl） </td><td></td><td> ＆NBSP; </td></tr><tr><td>铜（I）溴化物（溴化亚铜） </td><td></td><td></td></tr><tr><td>氯化钙（ 氯化钙 ） </td><td></td><td></td></tr><tr><td>氯化钾（KCl） </td><td></td><td></td></tr><tr><td>氯化镁（MgCl 2的 ） </td><td></td><td></td></tr><tr><td>氯化钠（NaCl） </td><td></td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> 炔CuAAC *探头 </td><td></td><td></td></tr><tr><td> :TAMRA炔</td><td> Invitrogen公司</td><td> T10183 </td></tr><tr><td colspan="3"></td></tr><tr><td> 紧张炔探头SPAAC * </td><td></td><td></td></tr><tr><td> :TAMRA迪波炔</td><td> Invitrogen公司</td><td> C10410 </td></tr></tbody></table> *点击化学试剂和试剂盒的著名厂商包括Invitrogen公司，耶拿的生物科技公司，贝里联营公司，Sigma-Aldrich公司，格伦研究"化学工具，Baseclick。各种炔染料和靶向配体，可以发现在这些厂商的产品目录。

chemoselective modification of viral surfaces via bioorthogonal click chemistry

病毒颗粒的修改已收到的关注显着，其影响的基因治疗，溶瘤应用和疫苗开发的巨大潜力。1,2,3修改病毒的表面，其中大多是遗传学的基础，当前的方法往往受到衰减生产病毒，传染性和细胞转导。4,5使用化学选择性的点击化学，我们已经开发出一个简单的替代方法回避这些问题，同时保持高度灵活和方便。1,2 本议定书的目标是展示使用bioorthogonal点击化学修改的5型腺病毒颗粒表面的成效。可用于同时治疗1或分析，2,6这两个步骤的过程，作为靶向分子，染料或其他分子的化学选择性结扎允许到蛋白质预先标记叠标签。这种方法的三个主要优点是:（1）代谢标记表明几乎没有病毒健身的影响，1,7（2）广泛的效应配体可以利用，（3）它是非常快速，可靠和易于访问。1,2,7 在此过程的第一步，腺病毒颗粒生产轴承要么azidohomoalanine（AHA，蛋氨酸的替代）或不自然的糖O型联的 N-azidoacetylglucosamine（ 邻 GlcNAz），它们都含有叠氮化物（氮3）功能组。净化后的叠改性病毒颗粒，包含:TAMRA荧光基炔探头结扎预标记的蛋白质或糖蛋白在化学选择性的方式。最后，进行SDS-PAGE分析证明到病毒衣壳蛋白探头成功结扎。阿哈纳入标记所有病毒衣壳蛋白（Hexon蛋白，聚氯和光纤），而O型 GlcNAz只标签纤维掺入结果。 在这个不断发展的领域，已成功开发出多个叠 - 炔结扎方法，但是只有两个我们已经找到了，是最方便的证明本 - 促进应变叠氮化物 - 炔环加成（SPAAC）和铜催化的叠炔环加成（CuAAC）在无氧气氛下。

Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

腺病毒颗粒设计，含有非天然氨基酸类似物azidohomoalanine或叠氮糖<em&gt;Ø</em&gt; GlcNAz。每叠组chemoselectively结扎通过"点击"化学反应病毒表面改性的一种手段。

通过Bioorthogonal点击化学化学选择性病毒表面改性

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

chemoselective-modification-viral-surfaces-via-bioorthogonal-click

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

24.0K 次观看.  Stony Brook University. 腺病毒颗粒设计，含有非天然氨基酸类似物azidohomoalanine或叠氮糖Ø GlcNAz。每叠组chemoselectively结扎通过"点击"化学反应病毒表面改性的一种手段。

视频: Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

一个<em&gt;体外</em&gt; Caseum结合测定，预测药物在渗透结核病变

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...