Name: highly sensitive assay for measurement of arenavirus-cell attachment
Uploaded: 2016-03-02T14:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment at JoVE.com

我们感谢马库斯·塔利，弥敦道罗伊，克里斯托弗·齐格勒，艾米莉布鲁斯，便雅悯王为通过赠款T32 AI055402（JK），R21 AI088059（JB），以及P20RR021905有益的探讨和美国国立卫生研究院支持这些研究（JB） 。

注意:这是非常关键的，所描述的协议，采用严格的预PCR技术（见28关于如何建立一个-PCR实验室前和如何使用好-PCR技术前进行实验的精彩概述）进行。至关重要的是，所有的试剂和仪器都是免费的含有定量PCR靶序列扩增的DNA，并有适当的控制到位，以检测这种污染。该协议的概述示于图1。 1.准备在48孔板媒体和种子细胞<ol><li>通过加入50ml制备含有10％胎牛血清，1％青霉素 - 链霉素500毫升完整的Dulbecco改良Eagle培养基中（DMEM）中，和1％的HEPES（N-2-羟乙基-N-2-乙烷磺酸）缓冲液的热灭活胎牛血清和5ml每一个100X青霉素 - 链霉素和100X HEPES缓冲溶液到500ml瓶中的DMEM组成。该试剂可以被存储在476℃持续几个月。 </li><li>种子2.5×10 4个 Vero E6细胞每孔在500μl的完全DMEM的最终体积的48孔组织培养板。孵育在37℃的板在含5％CO 2的10至18小时的湿润培养箱。 <ol><li>在工作​​日的最后种子板块。测试在任一复制或三孔每个病毒样本。 注意:虽然用于本测定基于48孔平台被描述，但应该有可能向下使用缩放测定在96-或384-孔板中。 </li></ol></li></ol> 2，开展病毒细胞附着注意:在这个协议中使用的病毒，JUNV应变偷拍＃1（C＃1）中，已成功地用作用于预防JUNV疾病29的减毒活疫苗。 JUNV C＃1，从通过在体内和体外连续传代的毒力JUNV应变XJ衍生和由12个氨基酸30亲XJ菌株不同。因为...ËJUNV C＃是1级生物安全（BSL）-2级病原体，本节介绍的工作，必须使用适当的BSL-2实践31进行。这包括使用个人防护装备（ 例如眼睛保护，实验室外套，双层手套），II级生物安全柜，并确保所有人员都收到了机构培训，并安全地处理这个有机体所需的批准。 <ol><li>稀释JUNV C＃1样品（多个）进行测试，以所希望的PFU就能或者在冰冷完全DMEM焦点形成单位并存储在冰上直到准备添加到每个孔中。 注意:给导电与病毒在完全DMEM稀释结合研究另一种选择，使用含有PBS的基本培养基具有降低的血清浓度（2％）和合适的缓冲液来建立血清是否可能会导致与病毒附着干扰。替代病毒类似附件协议已报道没有bicarbo使用RPMI 1640培养基内特含有0.2％牛血清白蛋白，10mM的（2-（吗啉代）乙磺酸）和10mM HEPES，pH 6.8的32。这种结合媒体可能优于防止当介质从培养箱中的CO 2 -environment除去可能发生的pH变化。 <ol><li>接种与PFU就能井从200至2,000，以限制非生产性附着事件。正如图2所示，该测定能够利用少至20 PFU（或多达2×10 6 PFU）检测附件。 </li><li>创建病毒的预混接种到细胞上。每种病毒的样本将在每孔50μl的体积接种到重复的孔中。因此，如果每孔病毒的2000 PFU就能增加是需要的，稀释4800 PFU就能为120微升冰的总体积冷完成DMEM中。 <ol><li>为了控制用于测定的特异性，使用一对匹配的中国仓鼠卵巢细胞系的，要么不表达TFR1（TRVb）或EXPRESS hTfR1（TRVb-1），为JUNV附着到这些细胞要么应不分别13,33减少或。另外，治疗用蛋白酶的细胞，如蛋白酶K，以防止沙粒病毒附件34或入境35。可溶性hTfR1或单克隆抗体ch128.1，这是具体 ​​的hTfR1，也可以考虑，因为它们是公知的方框JUNV的进入细胞13,36。此外，免费的甘露糖14或特定JUNV中和抗体37颗粒孵化预处理细胞可以抑制病毒进入。 </li><li>但是，请注意，这些试剂后者和方法，尽管拦截JUNV项，尚未得到确认阻止的附件，虽然这是一个可能的解释。此处所描述的协议可用于正式确定这些各种方法的影响病毒体附着。 </li></ol></li></ol></li><li>从37℃培养箱中取出盘子和放置或者在4℃冰箱或在冰上15分钟以抑制铺板的细胞来进行细胞内吞作用的能力。 </li><li>接着，吸出从每个完全DMEM井，并使用100μl的冰冷的PBS（磷酸盐缓冲盐水），PH值7.4的迅速洗每个孔两次。然后加入50微升预稀释的病毒样品（在步骤2.1中制备）向适当孔中。 </li><li>包裹板保鲜膜，并立即将要么回到冰上或4℃冰箱中1〜1.5小时，以允许进行病毒附件。保持板，洗涤缓冲液，和病毒的样品冷在4℃贯穿此步骤。 </li><li>在4℃温育完成后，吸出病毒接种物，并用200微升冰冷的PBS洗每个孔3次。 </li></ol> 3.病毒RNA提取<ol><li>通过使用商业试剂盒（ 例如 ，的RNeasy Mini试剂盒）雅从JUNV C＃纯化病毒RNA附着在单层1颗粒鼎制造商的协议。具体来说，按照上中的RNeasy Mini手册（ 第 4版，2006年4月）下面列出的修改页面23-28"使用旋转技术的协议动物细胞的总RNA净化"。 <ol><li>裂解如通过直接添加350微升裂解缓冲液RLT中向每个孔朝向步骤1B中的细胞。混合缓冲几次，以确保细胞溶解。注意，细胞容易在该缓冲溶解和完全裂解可通过倒置显微镜下观察孔进行验证。 </li><li>转移得到的细胞裂解物的试剂盒，柱（步骤3a）中，并按照协议的其余部分所描述的。在这一点上，该病毒已被裂解缓冲液中和这样的协议的其余步​​骤可在II级生物安全柜的外面来完成所提供的试剂盒管不沾染感染性病毒。 </li><li>包括来自制造商的方案，这是一个ADDIT可选步骤9旋转柱的有理离心来消除缓冲液RPE的任何可能残留。 </li><li>在最后的步骤中，制造商的方案（步骤10），洗脱使用30微升无核酸的DDH 2 O的旋转柱纯化的RNA的在-80°C储存RNA在这一点上，或直接在定量RT-PCR检测使用。为了防止病毒纯化RNA样品降解核酸酶，使用无核酸试剂和设备，并保持解冻RNA应放在冰上。 注意:缓冲器RLT和RW1含有胍盐，不应与漂白剂混合。缓冲RW1含有乙醇。 </li></ol></li></ol>病毒基因组的4定量RT-PCR检测<ol><li>转换JUNV C＃1 S片段的RNA成cDNA用于随后的qPCR，若使病毒RNA（在步骤3.1.4生成）至RT在50微升的总体积。 <ol><li>要设置RT反应，首先创建一个包含各单位反应以下试剂预混液:6.75微升核酸-free DDH 2 O的，5微升2微米的库存的RT引物的S的2098-到2075-（5&#39;-AAGGGTTTAAAAATGGTAGCAGAC-3&#39;），这是向S段基因组RNA的NP区域，5微升的互补10X PCR缓冲液II，11微升的25mM MgCl 2溶液，1.25微升（62.5单位）逆转录酶，RNA酶抑制剂的1微升（0.4单位），以及10微升500μM的股票的dNTP的。 注意:在沙粒病毒复制的过程中，产生了4病毒S片段RNA种类:全长基因组RNA（vRNA节），全长基因组RNA（vcRNA）（即vRNA节的反向互补）和两个亚基因组mRNA的编码NP和GP基因，分别为。这里列出的RT引物是特异于仅S片段vRNA节，但不是vcRNA，NP的mRNA，或GP的mRNA。 </li><li>轻轻地混合RT预混液，然后分装40微升成单个0.2毫升PCR管。添加10微升病毒RNA至每个管。包括:i）一个无模板对照管（ 如:。添加无核酸酶DDH 2 O的40微升主混合物）和ii）一个没有RT酶对照的10微升（ 例如加10微升已知阳性病毒RNA样品到主混合物的40微升未接受任何逆转录酶）来筛选对RT试剂与含有病毒的靶序列扩增的DNA的污染。包括来自未感染的细胞与完整的RT主混合物，以控制在细胞RNA的存在下测定特异性提取的RNA。 </li><li>此外，包括已知的阳性病毒RNA样品充分预混，以验证对RT试剂的质量。注意，RT反应的体积可以根据需要降低到25微升。 </li></ol></li><li>若使RT管，以下面的循环条件:25℃，10分钟，48℃30分钟，和95℃5分钟。注意，样品可以通过加入4℃的步骤中保存在-20℃，在​​这一点上，或在热循环仪放置过夜。 </li><li> Perform的qPCR在25微升的总体积。 <ol><li>做出的qPCR主混合物通过混合下述:每2.5微升（9μM股票）的正向PCR引物S1937 +可1958+（5&#39;-CATGGAGGTCAAACAACTTCCT-3&#39;）和反向PCR引物小号2001-到1982-（5所述的qPCR探针小号1960+的&#39;-GCCTCCAGACATGGTTGTGA-3&#39;），2.5微升（2μM股票）至1975+（5&#39;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&#39;），和12.5微升2X通用qPCR主混合。注意，正向引物这里报告，这是具体 ​​的JUNV C＃1，由1的不同核苷酸从最初报告引物序列38，其靶向有毒JUNV应变Romero的。 </li><li>轻轻地混合溶液中，然后加入20微升到每个定量PCR好。添加5μl的每个RT反应，以适当的定量PCR孔中。进行定量PCR中一式三份进行测试各样品。请注意，如果需要的PCR反应体积可减小到低至12.5微升。 注意:与相关联的软件在定量PCR机将每个未知样品的值进行比较，以一系列编码JUNV C＃1 NP基因，这是定量PCR引物的靶质粒的标准稀释物产生JUNV C＃1 S片段的基因组RNA的绝对拷贝数和探头设置。的qPCR分析只能为落入标准曲线范围内的值提供定量。出于这个原因，包括质粒下列数量（一式三份的孔）:5，50，5×10 3，每5×10 5和5×10 7个拷贝良好。 </li></ol></li><li>加载的qPCR板进入热循环和运行以下反应条件:95℃，10分钟，15秒和60℃1分钟的95℃的40个循环。 <ol><li>收集和分析的数据，根据制造商的协议来确定每个样品中的病毒RNA的存在量。 </li></ol></li><li>要使用质粒含有焦油的qPCR新纯化后的样品建立标准曲线得到的序列，首先确定在该溶液中的质粒拷贝数。 <ol><li>计算出的质粒的分子量。如果整个质粒的序列是已知的，计算该使用以下公式:分子量=（[A * 312.2] + [G * 328.2] + [C * 288.2] + [T * 303.2] - 61.13）。请记住，包括对双链质粒的每条链中发现特定核苷酸的总数。 </li><li>如果该质粒的大小是已知的，但它的确切序列不是，计算出各双链DNA碱基对具有600兆瓦的假设下的兆瓦。 注:这里使用的将pCAGGS-JUNV C＃1 NP质粒具有的4.2×10 6兆瓦。 </li><li>一旦该质粒的MW已经确​​定，计算每微升多少副本存在于库存质粒溶液。 </li><li>先计算的质粒的总微克在该溶液中，然后将其转换为质粒的摩尔（ 例如 ，如果69微克质粒都包含在300微升的水中，然后6.9×10 -5μg质粒* 1摩尔/ 4.2×10 6微克质粒的=质粒1.6×10 -11摩尔），它可以被转换成拷贝数（ 例如质粒的1.6×10 -11摩尔* 6.022×10 23分子/摩尔= 9.8×10 12分子（或复印件））。 </li><li>通过该溶液的体积除以在质粒溶液质粒的总拷贝得到每微升份（ 例如 9.8×10 12拷贝/ 300微升= 3.28×10 10个拷贝）。 </li><li>适当稀释该溶液，使5微升体积可以装载到PCR板传递以建立标准曲线所需拷贝数的范围内。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+screen+for+genes+that+affect+Junin+virus+entry+uncovers+voltage-gated+calcium+channels+as+a+therapeutic+target.">siRNA screen for genes that affect Junin virus entry uncovers voltage-gated calcium channels as a therapeutic target.</a> Sci Transl Med. 5, 204ra131 (2013).</li><li>Ellenberg, P., Edreira, M., Scolaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+superinfection+of+Vero+cells+persistently+infected+with+Junin+virus.">Resistance to superinfection of Vero cells persistently infected with Junin virus.</a> Arch. Virol. 149, 507-522 (2004).</li><li>Brandenburg, B., 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=Imaging+poliovirus+entry+in+live+cells.">Imaging poliovirus entry in live cells.</a> PLoS Biol. 5, e183 (2007).</li><li>Petersen, J., 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+Major+Cellular+Sterol+Regulatory+Pathway+Is+Required+for+Andes+Virus+Infection.">The Major Cellular Sterol Regulatory Pathway Is Required for Andes Virus Infection.</a> PLoS pathogens. 10, e1003911 (2014).</li><li>Jonsson, N., Gullberg, M., Israelsson, S., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+studies+of+virus+interaction+at+the+host+cell+surface+using+enteroviruses+and+real-time+PCR.">A rapid and efficient method for studies of virus interaction at the host cell surface using enteroviruses and real-time PCR.</a> Virol J. 6, 217 (2009).</li><li>Jiang, J., 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=Hepatitis+C+virus+attachment+mediated+by+apolipoprotein+E+binding+to+cell+surface+heparan+sulfate.">Hepatitis C virus attachment mediated by apolipoprotein E binding to cell surface heparan sulfate.</a> J. Virol. 86, 7256-7267 (2012).</li><li>Shannon-Lowe, 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=Epstein-Barr+virus-induced+B-cell+transformation:+quantitating+events+from+virus+binding+to+cell+outgrowth.">Epstein-Barr virus-induced B-cell transformation: quantitating events from virus binding to cell outgrowth.</a> J. Gen. Virol. 86, 3009-3019 (2005).</li><li>Mifflin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+up+a+PCR+laboratory.">Setting up a PCR laboratory.</a> CSH Protoc. 2007, pdb top14 (2007).</li><li>Maiztegui, J. I., 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=Protective+efficacy+of+a+live+attenuated+vaccine+against+Argentine+hemorrhagic+fever.+AHF+Study+Group.">Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group.</a> J. Infect. Dis. 177, 277-283 (1998).</li><li>Goni, S. E., 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=Genomic+features+of+attenuated+Junin+virus+vaccine+strain+candidate.">Genomic features of attenuated Junin virus vaccine strain candidate.</a> Virus Genes. 32, 37-41 (2006).</li><li>Chosewood, L. C., Wilson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in microbiological and biomedical laboratories. , (2009).</li><li>White, J., Matlin, K., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fusion+by+Semliki+Forest,+influenza,+and+vesicular+stomatitis+viruses.">Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses.</a> J. Cell Biol. 89, 674-679 (1981).</li><li>McGraw, T. E., Greenfield, L., Maxfield, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+the+human+transferrin+receptor+cDNA+in+Chinese+hamster+ovary+cells+deficient+in+endogenous+transferrin+receptor.">Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.</a> J. Cell Biol. 105, 207-214 (1987).</li><li>Borrow, P., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+lymphocytic+choriomeningitis+virus-binding+protein(s):+a+candidate+cellular+receptor+for+the+virus.">Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus.</a> J. Virol. 66, 7270-7281 (1992).</li><li>Rojek, J. M., Spiropoulou, C. F., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cellular+receptors+for+the+South+American+hemorrhagic+fever+viruses+Junin,+Guanarito,+and+Machupo.">Characterization of the cellular receptors for the South American hemorrhagic fever viruses Junin, Guanarito, and Machupo.</a> 346. , 476-491 (2006).</li><li>Helguera, G., 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=An+antibody+recognizing+the+apical+domain+of+human+transferrin+receptor+1+efficiently+inhibits+the+entry+of+all+new+world+hemorrhagic+Fever+arenaviruses.">An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses.</a> J. Virol. 86, 4024-4028 (2012).</li><li>Sanchez, 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=Junin+virus+monoclonal+antibodies:+characterization+and+cross-reactivity+with+other+arenaviruses.">Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses.</a> J. Gen. Virol. 70, 1125-1132 (1989).</li><li>Trombley, A. 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=Comprehensive+panel+of+real-time+TaqMan+polymerase+chain+reaction+assays+for+detection+and+absolute+quantification+of+filoviruses,+arenaviruses,+and+New+World+hantaviruses.">Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.</a> Am. J. Trop. Med. Hyg. 82, 954-960 (2010).</li><li>Helenius, A., Marsh, M., White, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Semliki+forest+virus+penetration+by+lysosomotropic+weak+bases.">Inhibition of Semliki forest virus penetration by lysosomotropic weak bases.</a> J. Gen. Virol. 58 (Pt 1), 47-61 (1982).</li><li>Nour, A. M., Li, Y., Wolenski, J., Modis, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+membrane+fusion+and+nucleocapsid+delivery+into+the+cytoplasm+are+distinct+events+in+some+flaviviruses.">Viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses.</a> PLoS pathogens. 9, e1003585 (2013).</li></ol>

作者宣称，他们没有竞争的经济利益。

我们描述的协议是简单的和健壮提供了以下关键的考虑因素观察。首先，严格预-PCR技术必须使用（参见第28一个很好的回顾）。至关重要的是，所有的试剂和仪器都是免费的含有定量PCR靶序列扩增的DNA，并有适当的控制到位，以检测这种污染。第二，对于协议，病毒，细胞和介质的病毒 - 细胞附着部分必须在4℃下保持，以防止表面结合的病毒粒子的内吞作用。第三，收集用于RNA提取的细?...

沙粒病毒是那些主要保持在啮齿类动物中自然1-3笼罩，单链RNA病毒家族。虽然这些病毒通常建立在啮齿动物水库1,2无症状，持续感染，可导致人类3严重疾病。重要的致病品种包括新世界胡宁病毒（JUNV）4和旧世界拉沙病毒5，分别是阿根廷出血热在南美和拉沙热的病原体在非洲。淋巴细胞脉络丛脑膜炎病毒（LCMV），家庭6的原型病毒，具有全球分销并负责多种疾病状态，包括在免疫活性个体6无菌性脑膜炎，在发育中的胎儿7严重的先天缺陷，或在免疫抑制高致死以下固体器官移植8,9个人。缺乏美国食品和药物管理局（FDA）批准的疫苗或打击这些病毒有效的抗病毒药物凸显迫切需要制定新的策略来治疗针对这些新兴/再出现的病原体。 的沙粒病毒基因组由两个单链RNA区段，所述大（L）（〜7.2 kb的）和小的（S）（〜3.6kb的）段10。而L区段编码的病毒聚合酶（L）和基质蛋白（Z） - 在S段编码病毒核蛋白（NP）和包膜糖蛋白（GP）。的L和S的基因组RNA区段（vRNAs）被打包成病毒颗粒10-12。沙粒病毒感染的最初步骤是病毒颗粒的附着通过病毒GP和其相应的宿主细胞受体的，为JUNV，包括人类的转铁蛋白受体1（hTfR1）13,14之间的相互作用对宿主细胞。以下附件，JUNV颗粒通过网格蛋白介导的内吞作用15取入细胞。颗粒然后投放到晚期内体，其中该隔室的低pH触发的GP 16,17的激活。一旦被激活，在GP-编码的融合肽插入到内体膜，病毒和内体膜之间引发熔合。成功融合结果在释放病毒粒子包装vRNAs的进入细胞质，在那里它们作为用于基因组复制和转录的模板。当产生病毒结构蛋白和vRNAs的足够数量时，新粒子组装和从感染细胞芽完成生命周期18。 为了促进新战略的发现到治疗目标致病沙粒病毒，我们集团一直专注于主机因素是主机的病毒的繁殖至关重要，但可有可无的鉴定。在这种情况下，限定由这种宿主因素控制病毒生命周期的精确阶段（s）是必要的引导的抗病毒策略的发展。在此，我们描述了基于RT-PCR的定量（q）的测定法可用于测量用于识别选定的宿主的蛋白和/或抗病毒分子是否影响病毒进入19的该初始阶段的目的沙粒病毒-细胞附着。衡量沙粒病毒细胞附着替代方法有特色生物素标记20，荧光染料21，或者放射性同位素22的病毒体。缺点这些方法可包括用于大量输入病毒的，使用放射性的（ 例如高感染复数（MOI）的多重），和/或附加的处理步骤以制备输入病毒（ 例如浓缩和/或病毒的标签）的要求。特别是，使用高MOI的使得难以区分生产性与非生产性附着事件15,23。这里描述的基于定量RT-PCR的检测可以用尽可能少的20个牌匾检测附件事件明台病毒（PFU就能），换算成0.0004的MOI为48孔板。因此，测定的强灵敏度克服了高感染复数的要求以及任何病毒标签或浓度的步骤。此外，该测定可以是ⅰ）适于测量病毒体的内吞作用以及病毒基因组的释放入细胞质以下融合和ii）适于通过病毒特异性的qRT-PCR试剂的发展与其他病毒使用（例如，参见24-27）。

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>11965-118</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-163</td><td>100x</td></tr><tr><td>HEPES Buffer Solution</td><td>Life Technologies</td><td>15630-130</td><td>100x</td></tr><tr><td>PBS pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td>1x</td></tr><tr><td>Vero E6 cells</td><td>American Type Culture Collection</td><td>CRL-1586</td><td></td></tr><tr><td>Costar 48-well plate</td><td>Corning</td><td>3548</td><td></td></tr><tr><td>RNeasy mini kit</td><td>Qiagen</td><td>74106</td><td>do not mix buffers RLT or&amp;nbsp; RW1 with bleach</td></tr><tr><td>QIAshredder homogenizer</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>Taqman Universal PCR Master Mix</td><td>Life Technologies</td><td>4326614</td><td></td></tr><tr><td>Multiscribe Reverse Transcriptase (50U/&amp;micro;l)</td><td>Life Technologies</td><td>4311235</td><td></td></tr><tr><td>dNTP Mixture (10 mM; 2.5 mM each dNTP)</td><td>Life Technologies</td><td>N808-0260</td><td></td></tr><tr><td>GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution</td><td>Life Technologies</td><td>N808-0010</td><td></td></tr><tr><td>RNase Inhibitor (5000U/100 &amp;micro;l)</td><td>Life Technologies</td><td>N808-0119</td><td></td></tr><tr><td>RT primer 5&amp;rsquo;-AAGGGTTTAAAAAT&lt;br/&gt;GGTAGCAGAC-3&amp;rsquo;</td><td>Integrated DNA Technologies</td><td>250 nmol DNA oligo</td><td>standard desalting</td></tr><tr><td>Forward qPCR primer&amp;nbsp; 5&amp;rsquo;-CATGGAGGTCAAACAACTTCCT-3&amp;rsquo;</td><td>Life Technologies</td><td>4304971</td><td>standard desalting</td></tr><tr><td>Reverse qPCR primer 5&amp;rsquo;-GCCTCCAGACATGGTTGTGA-3&amp;rsquo;</td><td>Life Technologies</td><td>4304971</td><td>standard desalting</td></tr><tr><td>TaqMan Probe 5&amp;rsquo;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&amp;rsquo;</td><td>Life Technologies</td><td>4316033</td><td>HPLC purified</td></tr><tr><td>MicroAmp Fast Optical 96-well reaction plate (0.1 mL)</td><td>Life Technologies</td><td>4346906</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>Life Technologies</td><td>4376598</td><td>or other qPCR instrument</td></tr></tbody></table>

highly sensitive assay for measurement of arenavirus-cell attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

为了证明qPCR分析的灵敏度和动态范围，如在该协议的第4部分中所述编码JUNV C＃1（范围5-5×10 7个拷贝）的NP基因的质粒的已知量经受的qPCR。该测定是敏感的，能够可靠地检测作为目标核酸的5个拷贝（ 图2）少。此外，该测定的动态范围大并且， 如图2，可覆盖至少7日志模板。虽然未示出，我们已检测出核酸的多达5×10 9个拷贝。 </p...

Watch this Scientific Journal Video about Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment at JoVE.com

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

highly-sensitive-assay-for-measurement-of-arenavirus-cell-attachment

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.5K 次观看.  University of Vermont. The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

视频: Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

科研

免疫与感染

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

早期的病毒进入测定的鉴定和抗病毒化合物的评价

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 &#176;C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Here we present two protocols, one for measuring the specific growth rate and the other for the cell-binding ability of rotavirus using the plaque assay and RT-qPCR. These protocols are available for confirming the differences in phenotypes between rotavirus strains.

轮状病毒的特定生长速率及细胞结合能力的测定

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

AAV 滴定定量斑点印迹法及其在腺相关病毒组装活化蛋白功能评价中的应用

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

生物学

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells

Source: Tai, C., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp. (2015).
This video demonstrates an assay to evaluate antiviral agents that target specific steps of the viral entry into host cells and fusion with the endosome. The cells are incubated at a low temperature and then infected with HCV. The low temperature permits viral binding to host cells but inhibits internalization via endocytosis. The test antiviral compound is added, and the cells are incubated at physiological temperature. This shift to a higher temperature facilitates viral entry and allows assessment of the impact of the test compound on viral envelope fusion with endosomes.

评估测试化合物对宿主细胞中病毒进入和融合影响的测定

Note: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum

Start plating HT1080 cells on day one by diluting the cells to a concentration of 1 x 105 cells/mL in pre-warmed complete DMEM media.
Then, seed 100 microliters of cells, per well, into clear 96-well flat-bottomed plates to the concentration of 1 x 104 cells per well. Incubate the plate at 37 degrees Celsius with 5% carbon dioxide overnight for 16 to 22 hours.
On day two, generate serial dilutions of the serum samples of interest in 1.5-milliliter microcentrifuge tubes using pre-warmed complete DMEM.
To each tube with diluted serum samples, add 66 microliters of the 7.5 x 106 viral genome per microliter virus working solution. Mix the virus/serum dilutions by pipetting, and then, place the tubes containing the virus/serum mixtures in an incubator at 37 degrees Celsius with 5% carbon dioxide for 30 minutes, to allow potential neutralization to occur.
After 30 minutes, pipette 100 microliters of the virus/serum mixture to each well on the 96-well plate containing 1 x 104 cells per well, then, wrap the plate in a foil to place in an incubator at 37 degrees Celsius with 5% carbon dioxide overnight for 16 to 24 hours.
On day three, aspirate the media from the wells of the 96-well plate using a fume hood vacuum without disrupting the adhered cells, and add 50 microliters of 4% paraformaldehyde to each well. After wrapping the plate in foil, leave it for 10 minutes at room temperature.
Later, wash and aspirate the cells twice with 200 microliters of PBS at room temperature. After the second wash, pipette 200 microliters of pre-warmed PBS into each well, and wrap the plate in foil followed by incubation at 65 degrees Celsius for 90 minutes to denature endogenous alkaline phosphatase activity.
Following incubation, add 50 microliters of the freshly prepared, dissolved BCIP/NBT into each well, and incubate the wrapped plates at room temperature for 2 to 24 hours. Later, take photos of each well using a 4x objective lens in a light microscope camera, ensuring the consistent use of the same exposure, white balancing, and light settings for all assays.