Name: contact-free co-culture model for the study of innate immune cell activation during respiratory virus infection
Uploaded: 2021-02-28T15:00:00
Description: Watch this Scientific Journal Video about Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection at JoVE.com

我们要感谢新加坡国立大学耳鼻喉科和微生物学和免疫学系的研究人员，感谢他们在hNEC培养和病毒培养相关工作方面的帮助。我们还要感谢国立大学医院耳鼻喉科的外科医生和外科团队，感谢他们协助提供研究所需的细胞和血液样本。 这项研究由国家医学研究委员会资助，新加坡No。NMRC/CIRG/1458/2016（致德云·王）和MOH-OFYIRG19may-0007（致谭启森）。Kai Sen Tan是2019年欧洲过敏和临床免疫学（EAACI）研究奖学金支持的获得者。

注：有关此协议中使用的介质配方，请参阅 表 1 。 注意：已发现在12孔透孔上生长的hNECS可生长成更优化的厚度，使可溶性因子在感染流感病毒时容易到达基底室。因此，建议使用12孔大小的转孔进行共培养。
1. 建立3T3供料层
<ol><li>从冻结的库存中建立<ol>
<li>从冷冻储液中解冻NIH / 3T3成纤维细胞的冷冻管。将冷冻管的内容物加入2 mL完全Dulbecco的最小必需培养?...

<ol>
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S., 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=RNA+sequencing+of+H3N2+influenza+virus-infected+human+nasal+epithelial+cells+from+multiple+subjects+reveals+molecular+pathways+associated+with+tissue+injury+and+complications.">RNA sequencing of H3N2 influenza virus-infected human nasal epithelial cells from multiple subjects reveals molecular pathways associated with tissue injury and complications.</a> Cells. 8 (9), 986 (2019).</li><li>Kim, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+lipopolysaccharide+on+diesel+exhaust+particle-induced+junctional+dysfunction+in+primary+human+nasal+epithelial+cells.">Effect of lipopolysaccharide on diesel exhaust particle-induced junctional dysfunction in primary human nasal epithelial cells.</a> Environmental Pollution. 248, 736-742 (2019).</li><li>Tan, K., 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=In+vitro+model+of+fully+differentiated+human+nasal+epithelial+cells+infected+with+rhinovirus+reveals+epithelium-initiated+immune+responses.">In vitro model of fully differentiated human nasal epithelial cells infected with rhinovirus reveals epithelium-initiated immune responses.</a> Journal of Infectious Diseases. 217 (6), 906-915 (2018).</li><li>Tan, K. S., 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=Comparative+transcriptomic+and+metagenomic+analyses+of+influenza+virus-infected+nasal+epithelial+cells+from+multiple+individuals+reveal+specific+nasal-initiated+signatures.">Comparative transcriptomic and metagenomic analyses of influenza virus-infected nasal epithelial cells from multiple individuals reveal specific nasal-initiated signatures.</a> Frontiers in Microbiology. 9, 2685 (2018).</li><li>Cytokine /Chemokine Panel II 96 Well Plate Assay. Millipore Corporation Available from: <a target="_blank" href="https://www.merckmillipore.com/SG/en/product/MILLIPLEX-MAP-Human-Cytokine-Chemokine-Magnetic-Bead-Panel-II-Premixed-23-Plex-Immunology-Multiplex-Assay">https://www.merckmillipore.com/SG/en/product/MILLIPLEX-MAP-Human-Cytokine-Chemokine-Magnetic-Bead-Panel-II-Premixed-23-Plex-Immunology-Multiplex-Assay</a> (2020)</li><li>Gamage, A. 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=Infection+of+human+nasal+epithelial+cells+with+SARS-CoV-2+and+a+382-nt+deletion+isolate+lacking+ORF8+reveals+similar+viral+kinetics+and+host+transcriptional+profiles.">Infection of human nasal epithelial cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.</a> PLoS Pathogens. 16 (12), 1009130 (2020).</li><li>Zhao, X. N., 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+use+of+nasal+epithelial+stem/progenitor+cells+to+produce+functioning+ciliated+cells+in+vitro.">The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro.</a> American Journal of Rhinology &amp; Allergy. 26 (5), 345-350 (2012).</li></ol>

针对病毒的先天免疫反应是抗病毒管理中一个尚未得到充分研究的领域。气道上皮细胞和先天免疫细胞协同工作以抑制感染期间的病毒复制，此外，如果病毒载量未受到控制，则作为过度反应的决定因素12，13，17。然而，开发用于研究上皮 - 先天免疫串扰的相关人类模型以研究先天免疫细胞的激活以赋予适当的抗病毒反应仍然...

呼吸道病毒可能是最普遍的病原体之一，造成严重的医疗保健和经济负担。从每年定期爆发的新流行毒株（如H1N1、H5N1、H3N2、MERS、COVID-19）到季节性流感毒株，病毒对公共卫生构成持续威胁。虽然疫苗构成了应对这些全球公共卫生挑战的主要部分，但令人清醒的是，这些对策只是响应1，2。此外，在新的传染性菌株的出现与其疫苗的成功开发之间出现延迟是不可避免的3，导致遏制病毒传播的现有措施非常有限。
这些拖延因给社会造成的经济和社会代价而进一步凸显。仅季节性流感每年就造成约80亿美元的间接成本、32亿美元的医疗费用和3.63万人死亡4。这是在考虑资助疫苗开发所需的研究成本之前。流行病的暴发对社会的影响甚至更加严重，而全球化的速度每年都在加快，严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）的出现和迅速传播造成的全球破坏就证明了这一点， 5，6，7。
最近的研究表明，具有更多活化先天性T细胞群的感染患者往往具有更好的疾病结果8，9，10。此外，先天性T细胞群被分类为多个亚组：粘膜相关不变T（MAIT）细胞，Vδ1 γδ T细胞，Vδ2 γδ T细胞和自然杀伤T（NKT）细胞。这些先天性T细胞亚群在其群体内也表现出异质性，增加了参与先天免疫应答的细胞群11之间相互作用的复杂性。因此，激活这些先天性T细胞的机制和先天性T细胞特定亚群的知识可能提供不同的研究途径，以减少这些病毒对人类宿主的感染作用，特别是在疫苗开发期间。
受流感感染的上皮细胞产生迅速激活先天性T细胞的因子12，13，14。基于这一发现，这种非接触式空气 - 液体界面（ALI）共培养模型旨在模拟感染的鼻上皮层和PBMCs在早期感染期间的早期化学相互作用（由受感染的上皮层释放的可溶性因子介导）。鼻上皮层（在膜插入物上培养）和PBMC（在下面的腔室中）之间的物理分离以及上皮完整性可防止PBMCs被病毒直接感染，从而可以详细研究上皮衍生的可溶性因子对PBMCs的影响。因此，可以进一步研究所确定的因素在诱导适当的先天性T细胞群方面的治疗潜力，从而可以预防流感感染。因此，本文详细介绍了建立共培养的方法，用于研究上皮衍生的可溶性因子的先天性T细胞活化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Ethylenediaminetetraacetic acid (EDTA), pH 8.0, RNase-free</td>
			<td>Thermofisher</td>
			<td>AM9260G</td>
			<td>0.5M EDTA</td>
		</tr>
		<tr>
			<td>1.5 mL SafeLock Tubes</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td>1.5mL Centrifuge Tube</td>
		</tr>
		<tr>
			<td>10 mL K3EDTA Vacutainer Tubes</td>
			<td>BD</td>
			<td>366643</td>
			<td>10mL Blood Collection Tubes</td>
		</tr>
		<tr>
			<td>10x dPBS</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Vivantis</td>
			<td>PC0711</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Plate</td>
			<td>Corning&#160;</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Transwell Insert</td>
			<td>Corning&#160;</td>
			<td>3460</td>
			<td>membrane insert</td>
		</tr>
		<tr>
			<td>1x FACS Lysing Solution</td>
			<td>BD</td>
			<td>349202</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL SafeLock Tubes</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td>2 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>24-well Plate</td>
			<td>Corning&#160;</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Transwell Insert</td>
			<td>Corning&#160;</td>
			<td>3470</td>
			<td></td>
		</tr>
		<tr>
			<td>3% Acetic Acid with Methylene Blue</td>
			<td>STEMCELL Technologies</td>
			<td>07060</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;,5-triiodo-l-thyronine</td>
			<td>Sigma</td>
			<td>T-074</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldehyde Solution w 15% Methanol as Stabilizer in H2O</td>
			<td>Sigma</td>
			<td>533998</td>
			<td></td>
		</tr>
		<tr>
			<td>5810R Centrifuge</td>
			<td>Eppendorf</td>
			<td>5811000320</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene tubes (flow tubes)</td>
			<td>BD</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Cell Strainer</td>
			<td>Corning&#160;</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>A-4-62 Rotor</td>
			<td>Eppendorf</td>
			<td>5810709008</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>Cell Dissociation Reagent</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Avicel CL-611</td>
			<td>FMC Biopolymer</td>
			<td>NA</td>
			<td>Liquid Overlay</td>
		</tr>
		<tr>
			<td>Bio-Plex Manager 6.2 Standard Software</td>
			<td>Bio-Rad Laboratories, Inc</td>
			<td>171STND01</td>
			<td>Multiplex Manager Software</td>
		</tr>
		<tr>
			<td>Butterfly Needle 21 G</td>
			<td>BD</td>
			<td>367287</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet&#160;</td>
			<td>Merck</td>
			<td>C6158</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm Solution</td>
			<td>BD</td>
			<td>554722</td>
			<td>Fixation and Permeabilization Solution</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma</td>
			<td>D4693</td>
			<td>Neutral Protease</td>
		</tr>
		<tr>
			<td>DMEM/High Glucose</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Nutrient Mixture F-12</td>
			<td>Gibco-Invitrogen</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Promega</td>
			<td>U1515</td>
			<td>dNTP Mix</td>
		</tr>
		<tr>
			<td>EMEM (w L-Glutamine)</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM voltohmmeter device</td>
			<td>WPI, Sarasota, FL, USA</td>
			<td>300523</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Lysing Solution</td>
			<td>BD</td>
			<td>349202</td>
			<td>1x Lysing Solution</td>
		</tr>
		<tr>
			<td>Falcon tube 15 mL</td>
			<td>CellStar</td>
			<td>188271</td>
			<td>15 mL tube</td>
		</tr>
		<tr>
			<td>Falcon tube 50 mL</td>
			<td>CellStar</td>
			<td>227261</td>
			<td>50 mL Tube</td>
		</tr>
		<tr>
			<td>Fast Start Essential DNA Probes Master</td>
			<td>Roche</td>
			<td>6402682001</td>
			<td>qPCR Master Mix</td>
		</tr>
		<tr>
			<td>Ficoll Paque Premium</td>
			<td>Research Instruments</td>
			<td>17544203</td>
			<td>Density Gradient Media</td>
		</tr>
		<tr>
			<td>H3N2 (A/Aichi/2/1968)&#160;</td>
			<td>ATCC</td>
			<td>VR547</td>
			<td></td>
		</tr>
		<tr>
			<td>H3N2 M1 Forward Primer Sequence</td>
			<td>Sigma</td>
			<td colspan="2">5&#39;- ATGGTTCTGGCCAGCACTAC-3&#39;</td>
		</tr>
		<tr>
			<td>H3N2 M1 Reverse Primer Sequence</td>
			<td>Sigma</td>
			<td colspan="2">5&#39;- ATCTGCACCCCCATTCGTTT-3&#39;</td>
		</tr>
		<tr>
			<td>H3N2 NS1 Forward Primer Sequence</td>
			<td>Sigma</td>
			<td colspan="2">5&#39;- ACCCGTGTTGGAAAGCAGAT-3&#39;</td>
		</tr>
		<tr>
			<td>H3N2 NS1 Reverse Primer Sequence</td>
			<td>Sigma</td>
			<td colspan="2">5&#39;- CCTCTTCGGTGAAAGCCCTT-3&#39;</td>
		</tr>
		<tr>
			<td>Heat Inactivated Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>hNESPCs</td>
			<td>Human Donors</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Epithelial Growth Factor</td>
			<td>Gibco-Invitrogen</td>
			<td>PHG0314</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>STEMCELL Techonologies</td>
			<td>7925</td>
			<td>Collected from nasal biopsies during septal deviation surgeries</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I3536</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightcycler 96</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td>qPCR Instrument</td>
		</tr>
		<tr>
			<td>Live/DEAD Blue Cell Stain Kit *for UV Excitation</td>
			<td>Thermofisher</td>
			<td>L23105</td>
			<td>Viability Stain</td>
		</tr>
		<tr>
			<td>MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel II - Premixed 23 Plex</td>
			<td>Merck Pte Ltd</td>
			<td>HCP2MAG-62K-PX23</td>
			<td>Immunology Multiplex Assay</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Sigma</td>
			<td>M4287</td>
			<td></td>
		</tr>
		<tr>
			<td>M-MLV 5x Buffer</td>
			<td>Promega</td>
			<td>M1705</td>
			<td>RT-PCR 5x Buffer</td>
		</tr>
		<tr>
			<td>M-MLV Reverse Transcriptase</td>
			<td>Promega</td>
			<td>M1706</td>
			<td>Reverse Transcriptase</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco-Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH/3T3</td>
			<td>ATCC</td>
			<td>CRL1658</td>
			<td></td>
		</tr>
		<tr>
			<td>Perm/Wash Buffer</td>
			<td>BD</td>
			<td>554723</td>
			<td>Permeabilization Wash Buffer</td>
		</tr>
		<tr>
			<td>PneumaCult-ALI 10x Supplement</td>
			<td>STEMCELL Techonologies</td>
			<td>5001</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Basal Medium</td>
			<td>STEMCELL Techonologies</td>
			<td>5001</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Maintenance Supplement (100x)</td>
			<td>STEMCELL Techonologies</td>
			<td>5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Primers</td>
			<td>Promega</td>
			<td>C1181</td>
			<td>Random Primers</td>
		</tr>
		<tr>
			<td>Recombinant Rnasin Rnase Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
			<td>RNase Inhibitor</td>
		</tr>
		<tr>
			<td>RNA Lysis Buffer</td>
			<td>Qiagen</td>
			<td>Part of 52904</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 (w L-Glutamine)</td>
			<td>ATCC</td>
			<td>30-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>STX2 electrodes</td>
			<td>WPI, Sarasota, FL, USA</td>
			<td>STX2</td>
			<td>Electrode</td>
		</tr>
		<tr>
			<td>T25 Flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>TPCK Trypsin</td>
			<td>Sigma</td>
			<td>T1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Hyclone</td>
			<td>SV30084.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Viral RNA Extraction Kit</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td>Viral RNA Extraction Kit</td>
		</tr>
		<tr>
			<td>V-Shaped 96-well Plate</td>
			<td>Corning</td>
			<td>3894</td>
			<td></td>
		</tr>
	</tbody>
</table>

contact-free co-culture model for the study of innate immune cell activation during respiratory virus infection

在病毒感染期间，鼻上皮层和先天免疫细胞之间的早期相互作用仍然是一个探索不足的领域。先天免疫信号传导在病毒感染中的重要性已大大增加，因为表现出高先天性T细胞活化的呼吸道感染患者显示出更好的疾病结果。因此，剖析这些早期先天免疫相互作用可以阐明控制它们的过程，并可能促进潜在的治疗靶点和策略的发展，以抑制甚至预防病毒感染的早期进展。该协议详细介绍了一种多功能模型，可用于研究病毒感染的气道上皮细胞分泌的因子中先天免疫细胞的早期串扰，相互作用和激活。使用H3N2流感病毒（A/ Aichi / 2 / 1968）作为代表性病毒模型，使用流式细胞术分析了共培养外周血单核细胞（PBMC）的先天细胞活化，以研究由上皮释放的可溶性因子激活的细胞亚群以响应病毒感染。结果验证了分化细胞亚群的门控策略，揭示了PBMCs活化群体及其与对照和感染上皮的串扰之间的明显差异。然后可以进一步分析激活的亚群以确定其功能以及细胞特异性的分子变化。这种串扰研究的结果可能会发现对激活重要先天细胞群很重要的因素，这些因素有利于控制和抑制病毒感染的进展。此外，这些因素可以普遍适用于不同的病毒性疾病，特别是新出现的病毒，以减轻这些病毒首次在幼稚人群中传播时的影响。

虽然传统的T细胞构成了针对病毒感染的适应性免疫反应的主要库，以促进病毒清除，但先天性T细胞群在更广泛的谱系中起作用，以抑制病毒载量，以便在后期有效清除。因此，该方案专门创造了一种强大的条件来研究流感感染后的先天性T细胞，它们的活化及其功能群体，而不需要来自同一供体的上皮和免疫细胞样本。该协议也可以应用于其他病毒，尽管它可能仅限于具?...

Watch this Scientific Journal Video about Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection at JoVE.com

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

该协议详细研究了病毒感染的鼻上皮细胞与先天细胞活化之间的早期相互作用。免疫细胞的单个亚群可以根据其对病毒感染的反应而激活来区分。然后可以进一步研究它们以确定它们对早期抗病毒反应的影响。

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

该协议旨在研究上皮细胞和一线先天免疫细胞的串扰，特别是先天性T细胞，这是我们感兴趣的。该技术使用来自人类来源的原代细胞，允许对与人类正在发生的事情相关的生理串扰进行个体研究。先天性T细胞与提高流感感染患者的生存率有关。了解它们如何被激活可以帮助开发抗流感和抗病毒疗法。该方法旨在识别可以激活先天性T细胞的因素，这可能有助于制定针对呼吸道病毒感染的预防措施。在零点，感染人鼻上皮细胞或HNEC。首先，通过将150微升1x胰蛋白酶EDTA加入膜插入物的顶室和350微升1x胰蛋白酶EDTA到代表性孔的基底室来计数细胞。将细胞在37摄氏度孵育10分钟或直到细胞从膜中分离。通过上下移液将细胞冲洗膜上，并将悬浮液收集在1.5毫升离心管中。加入200微升DMEM以淬灭胰蛋白酶活性。然后，通过台盼蓝染色计数细胞。根据每孔的细胞计数计算病毒所需的感染多重性或MOI。在冰上用完全的RPMI相应地稀释病毒储备。对于用于感染实验的其余孔，将一个1x DPBS加入膜插入物的顶端室中。在37摄氏度下孵育10分钟，然后取出1x DPBS。通过将插入物转移到新板上，将膜插入物转移到孔中加入完整RPMI的新板上，将膜插入物中的基础介质更改为完整的RPMI介质。将制备的病毒接种物加入膜插入物的顶腔中。在35摄氏度的5%二氧化碳气氛中孵育一小时，并从顶点室中取出病毒接种物。将膜插入物的基础介质更换为新鲜的RPMI培养基，并在35摄氏度的5%二氧化碳气氛中孵育24至72小时。从第零天开始，通过将PBMC悬浮液直接加入受感染HNEC每孔的基底室，在完整的RPMI培养基中接种所需数量的外周血单核细胞或PBMC。在37摄氏度下孵育24至48小时。为了收集顶端上清液，向每个顶端室加入1x DBPS，并在37摄氏度下孵育10分钟。然后，将DPBS收集在1.5毫升管中。在新的1.5毫升管中等分用于斑块测定的上清液，并立即在零下80摄氏度下冷冻储备液和等分试样。要收集RNA形式的HNEC，请将膜插入物转移到干净的孔中。将RNA裂解缓冲液加入顶端室，并在室温下孵育五分钟。将上清液收集在1.5毫升离心管中，并将其储存在零下80摄氏度直至RNA提取以进行分子分析。最后，通过用无菌移液器尖端的宽基部轻轻刮擦孔表面来收集PBMC，以清除可能粘附在孔表面的活化PBMCs。将含有PBMC的基础介质收集在两毫升离心管中。用300微升1x DPBS冲洗孔两次，并将洗涤液收集在同一个两毫升管中。离心两毫升管并将上清液收集在新鲜的两毫升管中，而不会干扰细胞沉淀。将吸出的上清液储存在零下80摄氏度，用于细胞因子和趋化因子分析。将剩余的细胞沉淀重悬于200微升1x DPBS中。该方案可用于研究流感感染后的先天性T细胞，而无需来自同一供体的上皮细胞和免疫细胞。此处显示了 HNESPC 在 3T3 馈线层上生长时的预期进展示例。这些用于在空气/液体界面培养物中进行分化，以获得具有功能性，纤毛形和高脚杯细胞的多层HNEC。使用HNEC，可以使用流式细胞术研究先天性T细胞活化。这些结果显示检测黏膜相关不变性T或MAIT细胞，Vδ 1，γδT细胞和自然杀伤T或NKT细胞。在涉及感染流感病毒的HNEC的共培养物中，这些细胞显着增加。然后，可以将这种设置应用于其他流感病毒株或先天免疫细胞群，以观察它们在与受感染的上皮细胞共培养条件下各自的激活。准确计算人鼻上皮细胞的数量以确保使用正确的感染多样性对于激活的最佳水平和时间非常重要。上皮细胞和先天免疫细胞之间的串扰越来越受到关注，这可以进一步应用于任何其他涉及急性或慢性呼吸道免疫学的方法。

Research

JoVE Journal

Immunology and Infection

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

科研

免疫与感染

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

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

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

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

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...