Name: merkel cell polyomavirus infection and detection
Uploaded: 2019-02-07T13:00:00
Description: Watch this Scientific Journal Video about Merkel Cell Polyomavirus Infection and Detection at JoVE.com

作者感谢 meenhard herlyn 博士 (wistar 研究所) 和 m. celeste simon 博士 (宾夕法尼亚大学) 提供试剂和技术支持。我们还感谢我们实验室的成员进行了有益的讨论。这项工作得到了国家卫生研究院 (r01ca187718、r01ca147778 和 R01CA187718)、nci 癌症中心支助赠款 (nci p30 c12016520) 和 penn cfar 奖 (p30 ai 045008) 的支持。

人类新生儿包皮是从宾州皮肤病研究中心获得的。成人人成纤维细胞是在手术后从废弃的正常皮肤中获得的。所有议定书都得到了宾夕法尼亚大学机构审查委员会的批准。
1. 人真皮成纤维细胞的分离
<ol><li>使用剪刀剪掉人类新生儿包皮的脂肪和皮下组织, 并将皮肤样本切成两半或四分之一。</li>
	<li>在 pbs 中将 10 mgml 消解酶 ii 中的5毫升组织在4°c 后补充抗生素抗真菌。</li>
	<...

<ol>
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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+Minimum+Replication+Origin+of+Merkel+Cell+Polyomavirus+Has+a+Unique+Large+T-Antigen+Loading+Architecture+and+Requires+Small+T-Antigen+Expression+for+Optimal+Replication.">The Minimum Replication Origin of Merkel Cell Polyomavirus Has a Unique Large T-Antigen Loading Architecture and Requires Small T-Antigen Expression for Optimal Replication.</a> Journal of Virology. 83 (23), 12118-12128 (2009).</li><li>Carter, J. 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=Identification+of+an+overprinting+gene+in+Merkel+cell+polyomavirus+provides+evolutionary+insight+into+the+birth+of+viral+genes.">Identification of an overprinting gene in Merkel cell polyomavirus provides evolutionary insight into the birth of viral genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (31), 12744-12749 (2013).</li><li>Seo, G. J., Chen, C. J., Sullivan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merkel+cell+polyomavirus+encodes+a+microRNA+with+the+ability+to+autoregulate+viral+gene+expression.">Merkel cell polyomavirus encodes a microRNA with the ability to autoregulate viral gene expression.</a> Virology. 383 (2), 183-187 (2009).</li><li>Theiss, J. M., 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=A+Comprehensive+Analysis+of+Replicating+Merkel+Cell+Polyomavirus+Genomes+Delineates+the+Viral+Transcription+Program+and+Suggests+a+Role+for+mcv-miR-M1+in+Episomal+Persistence.">A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence.</a> PLOS Pathogens. 11 (7), 1004974 (2015).</li><li>Schowalter, R. M., Pastrana, D. V., Buck, C. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Merkel+cell+polyomavirus+minor+capsid+protein.">The Merkel cell polyomavirus minor capsid protein.</a> PLOS Pathogens. 9 (8), 1003558 (2013).</li><li>Houben, R., Schrama, D., Becker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+of+Merkel+cell+carcinoma.">Molecular pathogenesis of Merkel cell carcinoma.</a> Experimental Dermatology. 18 (3), 193-198 (2009).</li><li>Chang, Y., Moore, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merkel+cell+carcinoma:+a+virus-induced+human+cancer.">Merkel cell carcinoma: a virus-induced human cancer.</a> Annual Review of Pathology. 7, 123-144 (2012).</li><li>Hodgson, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merkel+cell+carcinoma:+Changing+incidence+trends.">Merkel cell carcinoma: Changing incidence trends.</a> Journal of Surgical Oncology. 89 (1), 1-4 (2005).</li><li>Tolstov, Y. L., 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=Human+Merkel+cell+polyomavirus+infection+II.+MCV+is+a+common+human+infection+that+can+be+detected+by+conformational+capsid+epitope+immunoassays.">Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays.</a> International Journal of Cancer. 125 (6), 1250-1256 (2009).</li><li>Schowalter, R. M., Pastrana, D. V., Pumphrey, K. A., Moyer, A. L., Buck, C. 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E., Damania, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+viruses+and+innate+immunity.">Tumour viruses and innate immunity.</a> Philosophical Transactions of the Royal Society of London. 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上述方法包括从人体皮肤组织中分离真皮成纤维细胞、制备重组 mcpyv 病毒、培养细胞感染、免疫荧光染色和采用 hcr 技术的敏感 fish 方法。应该使研究人员能够分析 mcpyv 感染 27.在体外实现 mcpyv 感染的最关键步骤之一是生产高滴度病毒制剂。利用本文所述的重组 mcpyv 病毒的制备方案, 我们实现了每毫升的 10, 12个基因组拷贝的病毒滴度, 共 11,<sup ...

默克尔细胞多瘤病毒 (mcpyv) 是一种小型双链 dna 病毒, 与罕见但具有攻击性的皮肤癌, 默克尔细胞癌 (mcc)1,2。mcc 的死亡率, 约 33%, 超过黑色素瘤 3,4。mcpyv 的圆形基因组约为 5 kb 1,5 被非编码调控区 (ncrr) 一分为二, 进入早期和后期编码区域1。ncrr 包含病毒复制 (ori) 的病毒来源和病毒转录的双向启动子 6,7。早期区域编码肿瘤抗原蛋白称为大 t (lt), 小 t (st), 57kt, 替代 lt orf (alto), 以及自动调节 mirna1,8, 9, 10.后期区域编码衣壳蛋白 vp1 和 vp211,12,13。lt 和 st 是研究最多的 mcpyv 蛋白, 已被证明支持病毒 dna 复制和 mcpyv 诱导的肿瘤发生 5。mcpyv dna 在宿主基因组中的克隆整合, 在多达80% 的 mcc 中被观察到, 很可能是病毒阳性肿瘤发生 14,15的一个因果因素。
在过去20年中, mcc 的发病率增加了两倍16。无症状 mcpyv 感染在一般人群中也很普遍,为 17,18,19。随着 mcc 诊断数量的增加和 mcpyv 感染的高流行率, 有必要提高我们对病毒及其致癌潜力的认识。然而, mcpyv 生物学和致癌机制的许多方面仍然鲜为人知20。这主要是因为 mcpyv 在现有细胞系11、12、21、22、23以及直到最近的皮肤细胞中复制得很差, 直到最近, 还有能够支持 mcpyv 的皮肤细胞感染没有被发现 22。由于缺乏传播病毒的细胞培养系统, 充分研究 mcpyv 及其与宿主细胞相互作用的力学研究受到了阻碍。
我们发现, 从新生儿包皮中分离出的原代人真皮成纤维细胞 (hdf)在体外和体外24支持强大的 mcpyv 感染。通过本研究, 建立了 mcpyv24的第一个细胞培养感染模型。在该模型系统的基础上, 我们发现, 通过 wnt/β-儿茶素信号转导途径和其他生长因子诱导基质金属蛋白酶 (mmp) 基因刺激 mcpyv 感染。此外, 我们还发现, fda 批准的 mek 拮抗剂曲美替尼有效地抑制 mcpyv 感染5,25。通过这些研究, 我们还建立了一套隔离人真皮成纤维细胞 24,25的协议, 准备 mcpyv 病毒 11,12, 对人类真皮成纤维细胞进行 mcpyv 感染24,25和 mcpyv 蛋白的 if 染色26。此外, 我们还采用原位 dna 杂交链反应 (hcr) 技术27 , 开发了一种高度敏感的 fish 技术 (hcr-dna fish), 用于检测受感染的人类皮肤细胞中的 mcpyv dna。这些新方法将有助于研究 mcpyv 的感染周期以及细胞对 mcpyv 感染的反应。维持 mcpyv 感染的天然宿主水库细胞和引起 mcc 肿瘤的细胞仍然是未知的。我们在这篇手稿中描述的技术可以用来检查各种类型的人体细胞, 以确定储集细胞和 mcc 肿瘤的起源。我们已建立的方法, 如 hcr-dna fish, 也可用于检测其他 dna 肿瘤病毒和宿主细胞相互作用的特征。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal calf serum</td>
			<td style="width:185px;">HyClone</td>
			<td style="width:119px;">SH30071.03</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">GLUTAMAX I, 100X</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>L-Glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DPBS, no calcium, no magnesium</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">0.05% Trypsin-EDTA</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM/F12 medium</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Recombinant Human EGF Protein, CF</td>
			<td style="width:185px;">R&#38;D systems</td>
			<td>236-EG-200</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">CHIR99021</td>
			<td style="width:185px;">Cayman Chemical</td>
			<td>13122</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">CHIR99021</td>
			<td style="width:185px;">Sigma</td>
			<td>SML1046</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Collagenase type IV</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Dispase II</td>
			<td style="width:185px;">Roche</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Antibiotic-Antimycotic</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>15240-062</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM medium</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Alexa Fluor 594 goat anti-mouse IgG</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>A11032</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td>A11034</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">OptiPrep Density Gradient Medium</td>
			<td style="width:185px;">Sigma</td>
			<td>D1556</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">anti-MCPyV LT (CM2B4)</td>
			<td style="width:185px;">Santa Cruz</td>
			<td>sc-136172</td>
			<td>Lot # B2717</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">MCV VP1 rabbit</td>
			<td style="width:185px;"></td>
			<td>Rabbit polyclonal serum #10965</td>
			<td>https://home.ccr.cancer.gov/lco/BuckLabAntibodies.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Hygromycin</td>
			<td style="width:185px;">Roche</td>
			<td>10843555001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Basic Fibroblast Growth Factors (bFGF), Human Recombinant</td>
			<td style="width:185px;">Corning</td>
			<td>354060</td>
			<td>Store at -80 degree celsius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Benzonase Nuclease</td>
			<td style="width:185px;">Sigma</td>
			<td>E8263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Plasmid-Safe ATP-Dependent DNase</td>
			<td style="width:185px;">EPICENTRE</td>
			<td>E3101K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Probe hybridization buffer</td>
			<td style="width:185px;">Molecular technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">Probe wash buffer</td>
			<td style="width:185px;">Molecular technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Amplification buffer</td>
			<td style="width:185px;">Molecular technologies</td>
			<td style="width:119px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Alexa 594-labeled hairpins</td>
			<td style="width:185px;">Molecular technologies</td>
			<td>B4</td>
			<td>Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Triton X-100</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Quant-iT PicoGreen dsDNA Reagent</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">P7581</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">BamHI-HF</td>
			<td style="width:185px;">NEB</td>
			<td style="width:119px;">R3136</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Buffer PB</td>
			<td style="width:185px;">Qiagen</td>
			<td style="width:119px;">19066</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">blue miniprep spin column</td>
			<td style="width:185px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:185px;">Corning</td>
			<td style="width:119px;">352070</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">T4 ligase</td>
			<td style="width:185px;">NEB</td>
			<td style="width:119px;">M0202T</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagicMark XP</td>
			<td style="width:185px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">LC5602</td>
			<td></td>
		</tr>
	</tbody>
</table>

merkel cell polyomavirus infection and detection

默克尔细胞多瘤病毒 (mcpyv) 感染可导致默克尔细胞癌 (mcc), 这是一种极具攻击性的皮肤癌。由于缺乏适当的细胞培养模型, 充分研究 mcpyv 分子生物学和致癌机制的力学研究受到了阻碍。在这里, 我们描述了一组执行和检测人类皮肤细胞 mcpyv 感染的协议。这些协议描述了人真皮成纤维细胞的分离、重组 mcpyv 病毒的制备, 以及免疫荧光 (if) 染色和原位 dna 杂交链反应 (hcr) 检测病毒感染的方法, 这是一种高度敏感的方法。荧光原位杂交 (fish) 方法。有兴趣的研究人员可以对本文中的协议进行调整, 以确定支持 mcpyv 感染的其他细胞类型或细胞系。所描述的 fish 方法也可以用来检测受感染的人类皮肤中存在的低病毒 dna 水平。

本手稿中描述的协议允许隔离几乎同质的 hdf 群 (图 1)。免疫荧光染色显示, 使用本协议中描述的条件分离的人真皮细胞中, 几乎100% 的皮肤细胞对真皮成纤维细胞标记物、vimentin 和胶原蛋白 i24 进行了阳性染色, 但对人体呈阴性。包皮角质形成细胞标记 k14 (图 1)。图 2显示了使用重组 mcpyv ?...

Watch this Scientific Journal Video about Merkel Cell Polyomavirus Infection and Detection at JoVE.com

Merkel Cell Polyomavirus Infection and Detection

在这里, 我们提出了一个方案, 以感染原生真皮成纤维细胞与 mcpyv。该方案包括皮肤成纤维细胞的分离、mcpyv 病毒的制备、病毒感染、免疫荧光染色和荧光原位杂交。该协议可以扩展到描述 mcpyv-主机相互作用的特征, 并发现 mcpyv 注入的其他细胞类型。

默克尔细胞多瘤病毒感染与检测

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully ...

我们提出了默克尔细胞多母病毒感染的第一个细胞培养模型。该协议包括皮肤成纤维细胞的分离、MCPyV病毒感染的准备、免疫荧光染色和荧光原位杂交。该协议使得研究MCPyV感染周期和与宿主细胞的相互作用成为可能，同时避免了与病毒基因过度表达相关的伪影。首先，用一把剪刀修剪人类新生儿包皮的脂肪和皮下组织。将皮肤样本切成两半或四分之四。将组织放在5毫升10毫克每毫升非基II NPBS，辅以抗生素抗霉菌。一夜之间在4摄氏度下孵育。第二天，使用微切除钳子在10厘米的培养皿中小心地将表皮从皮肤层分离。将所得皮肤组织转移到含有每毫升2毫克的15毫升锥形管，不含FBS的介质中含有2毫克的胶原酶IV型，并辅以抗生素-抗原体。在37摄氏度的2氧化碳中孵育样品，定期摇晃4至6个小时，直到只保留几个宏观组织聚集体。在此之后，使用10毫升移液器将样品溶液上下大力移液10次，从真皮中释放单个细胞。在180克下离心5分钟，然后丢弃上一代。在补充的DMEM介质中镀块分离的细胞。在下午晚些时候或傍晚，将600万293TT28细胞种子放入含有补充DMEM培养基的10厘米培养皿中。在37摄氏度下孵育，二氧化碳含量为5%。第二天早上，确保细胞约50%的汇合，然后用66微升转染试剂转染细胞，12微克重合MPyV分离R17B DNA，8.4微克ST表达质粒pMtB，9.6微克LT表达质粒PADL。用5%的二氧化碳在37摄氏度下孵育细胞过夜。第二天，当转染细胞几乎汇合时，三叶草使细胞分裂，并转移到15厘米的培养皿中继续扩张。当该盘几乎汇合时，将细胞转移到三个新的15厘米的盘子中。当这些新菜几乎汇合时，收获文本协议中概述的细胞。在室温下180克离心5分钟。然后去除上一除以，并加入一个细胞体积的DPBS镁。加入25微摩尔硫酸铵，其次是0.5%Triton X-100，0.1%苯子酶，和0.1%的ATP依赖DNAse。混合好，在37摄氏度过夜孵育。第二天，在冰上冷却混合物15分钟。接下来，加入0.17体积的氯化钠。在冰上混合和孵育15分钟。在4摄氏度下在12000克下离心10分钟。如果上清液不清楚，轻轻反转管子并重复离心。在此之后，将上经器转移到新管中。将颗粒重新在 1 体积的 DPBS 中补充 0.8 摩尔氯化钠。在4摄氏度下在12000克下离心10分钟。如果上清液不清楚，轻轻反转管子并重复离心。将两种超级natant将和在4摄氏度下12000g再次离心10分钟。然后，使用移液器将碘异醇的梯度沉积到薄壁五毫升多聚体管中，如文本协议中所述。在准备好的碘黄醇梯度上加载3毫升的澄清性病毒的上清液。在 234000 g 和 16 摄氏度下使用 SW 55 Ti 转子的离心机，可保持 3.5 小时，确保将加速和减速设置为减速。超离心完成后，收集硅化管中的12个分数。维护和分离细胞后，在10毫升的补充DMEM和F12培养皿。将细胞溶液转移到15毫升管中。在180克时离心两分钟。丢弃上一提液，以每毫升2万至4万个细胞的细胞密度重新悬浮补充DMEM和F12培养基中的细胞。然后，种子200或400微升的细胞悬浮液补充1毫克每毫升胶原酶IV型到每井24或96井板，分别。从零下 80 摄氏度的冰柜中取出 MCPyV viron 库存，放在冰上解冻。每2500至5000个细胞每添加10亿个相当于MCPyV病毒的HIV基因。轻轻敲击板的一侧，在37摄氏度下孵育5%的二氧化碳。首先，在PBS中用4%PFA将细胞固定在盖滑上10分钟，然后用PBS洗涤盖滑两次，并在4摄氏度下用70%乙醇在四摄氏度下过夜，使细胞渗透。第二天，用探针水化缓冲液代替乙醇，在室温下孵育60分钟，使样品预杂交。在杂交前孵化结束前30分钟，在1：500稀释时稀释探针杂交溶液中的探针，并在45摄氏度下孵育。当孵育完成时，移液器上稀释探针混合物的大约10微升，用于每个盖滑的显微镜幻灯片。将每个盖滑单元侧向下放在其各自的杂交混合液滴上。使用自由量的橡胶水泥，密封每个盖的边缘和背面滑到其幻灯片。将幻灯片放在热块的平侧，加热至 94 摄氏度三分钟。之后，将滑梯转移到加湿室，并在45摄氏度下孵育。第二天，用钳子小心地剥去橡胶水泥。将盖板将细胞侧滑入 24 孔板的孔中，并在室温下用探头洗涤缓冲液清洗三次。在每个含盖滑的井中加入200微升扩增缓冲液，并在室温下孵育30至60分钟。同时，将两个标有寡核苷酸发夹的退火每个发夹在单独的PCR管中识别探针，加热到94摄氏度90秒，然后冷却到室温30分钟。在1：50的稀释下，将发夹混合在放大缓冲液中。接下来，将石蜡膜拉伸到12井板盖的打开面上，使表面发生放大反应。将发夹混合物的 50-100 微升液滴到每个盖滑的石蜡薄膜上。使用钳子，小心地从预放大溶液中去除每个盖滑，然后触摸边缘，使多孔的一次性擦拭干燥。将每个干燥的盖滑电池侧向下放置到放大液滴上。将板盖转移到加湿室中，在室温和黑暗中孵育过夜。第二天，将盖滑回24孔板的井中。将含有DAPI的5X SSCT加入到每毫升0.5微克的浓度中，并在室温下孵育一小时。在此之后，在室温下用 5X SSCT 清洗样品两次。将洗涤盖滑到显微镜幻灯片上，并使用倒置荧光显微镜分析细胞并成像样品。在这项研究中，一个几乎同质的人类皮肤成纤维细胞种群被隔离。免疫荧光染色表明，近100%的分离人类皮肤细胞被正面染色为皮肤纤维细胞标记物、维门丁和胶原蛋白，但人体外皮角细胞标记K14呈阴性。然后生成 MCPyV 病毒，并在可视化集中在梯度核心的 MCPyV 病毒带后，收集 500 微升分数，并执行 MCPyV qPCR 以识别峰值分数。此处显示了感染 MCPyV 的 HDF 的免疫荧光染色图像，其中 LT 阳性、VP1 阳性或 LT 和 VP1 阳性的细胞均被视为 MCPyV 感染阳性。超过 30% 的细胞为 LT 阳性，超过 10% 的细胞为 VP1 阳性。在受感染细胞中复制的MCPyV基因组使用HCR-DNA鱼和免疫荧光-HCR-DNA鱼检测。虽然 HCR-DNA FISH 揭示了复制工厂中存在的 MCPyV DNA 的定位，但免疫荧光-HCR-DNA FISH 方法允许在复制中心同时检测 MCPyV DNA 和 LT 蛋白的共定位。为了达到最佳的病毒感染效率，MCPyV集中在每微升至少2×10至8个病毒基因组中非常重要，因为碘氧醇对细胞有毒。在皮肤纤维细胞的MCPyV感染之后，可以进行许多分子生物分析，包括免疫荧光、免疫阻断、免疫位化和基于qPCR的测定。我们描述的感染系统使研究人员有可能研究MCPyV感染的阶段，如核贩运和病毒包装。MCPyV 是一种 BSL-2 病毒，但在实验室环境中达到的量中，其病理学潜力却很少为人所知。因此，研究人员应采取预防措施，避免接触病毒。

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

基于分子印迹的电容生物传感器超灵敏检测生物标志物

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...