Name: streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs
Uploaded: 2017-09-10T13:00:00
Description: Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

这项研究由 JDRF 1-2014-243-N, ADA 7-14-JF-07, NIH 5 P30 DK079638-05 飞行员 PJ, 和罗伯特和珍妮丝捷基金会资助。
我们感谢桑德拉和 Andrene 麦当劳的耐心招聘, 塞缪尔. 布鲁姆为技术援助, Dr. 乔治 Makedonas 的礼物抗体和控制 DNA 用于 PCR 优化。

1. 确定感兴趣的 T 细胞群体. 
 注意: 抗原特异增殖与细胞分裂染料 (如羧基琥珀酰亚胺酯, CFSE) 可用于分离 T 细胞的基础上, 其增殖反应抗原刺激。如果从 PBMCs 开始, 7 天的 体外 扩展应该足以识别抗原特异的 CFSE 低种群 12 。
<ol> <li> 将 PBMCs 1:1 与 MHC 匹配的进纸单元混合, 并使用5和 #181 的标签; M CFSE 细胞分裂染料. </li>
	<li> 板 2-2. 5 x 10 5 每个?...

<ol>
	<li>Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+development+and+the+CD4-CD8+lineage+decision.">T-cell development and the CD4-CD8 lineage decision.</a> Nat Rev Immunol. 2 (5), 309-322 (2002).</li><li>Singer, A., Adoro, S., Park, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+fate+and+intense+debate:+myths,+models+and+mechanisms+of+CD4-+versus+CD8-lineage+choice.">Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.</a> Nat Rev Immunol. 8 (10), 788-801 (2008).</li><li>Tubo, N. 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=Single+naive+CD4++T+cells+from+a+diverse+repertoire+produce+different+effector+cell+types+during+infection.">Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.</a> Cell. 153 (4), 785-796 (2013).</li><li>Sprouse, M. 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=Rapid+identification+and+expression+of+human+TCRs+in+retrogenic+mice.">Rapid identification and expression of human TCRs in retrogenic mice.</a> J Immunol Methods. , (2016).</li><li>Lennon, G. P., 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=T+cell+islet+accumulation+in+type+1+diabetes+is+a+tightly+regulated,+cell-autonomous+event.">T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event.</a> Immunity. 31 (4), 643-653 (2009).</li><li>Dash, P., 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=Paired+analysis+of+TCRalpha+and+TCRbeta+chains+at+the+single-cell+level+in+mice.">Paired analysis of TCRalpha and TCRbeta chains at the single-cell level in mice.</a> J Clin Invest. 121 (1), 288-295 (2011).</li><li>Kobayashi, 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=A+new+cloning+and+expression+system+yields+and+validates+TCRs+from+blood+lymphocytes+of+patients+with+cancer+within+10+days.">A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days.</a> Nat Med. 19 (11), 1542-1546 (2013).</li><li>Szymczak, A. 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=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat Biotechnol. 22 (5), 589-594 (2004).</li><li>Szymczak, A. L., Vignali, 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=Development+of+2A+peptide-based+strategies+in+the+design+of+multicistronic+vectors.">Development of 2A peptide-based strategies in the design of multicistronic vectors.</a> Expert Opin Biol Ther. 5 (5), 627-638 (2005).</li><li>Bettini, M. L., Bettini, M., Vignali, 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=T-cell+receptor+retrogenic+mice:+a+rapid,+flexible+alternative+to+T-cell+receptor+transgenic+mice.">T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice.</a> Immunology. 136 (3), 265-272 (2012).</li><li>Holst, J., Vignali, K. M., Burton, A. R., Vignali, 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=Rapid+analysis+of+T-cell+selection+in+vivo+using+T+cell-receptor+retrogenic+mice.">Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.</a> Nat Methods. 3 (3), 191-197 (2006).</li><li>Mannering, S. 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=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J Immunol Methods. 283 (1-2), 173-183 (2003).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Corthay, A., Nandakumar, K. S., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+percentage+of+peripheral+T+cells+with+two+different+T+cell+receptor+alpha-chains+and+of+their+potential+role+in+autoimmunity.">Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha-chains and of their potential role in autoimmunity.</a> J Autoimmun. 16 (4), 423-429 (2001).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> J Vis Exp. (113), (2016).</li><li>Bettini, M. L., Bettini, M., Nakayama, M., Guy, C. S., Vignali, 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=Generation+of+T+cell+receptor-retrogenic+mice:+improved+retroviral-mediated+stem+cell+gene+transfer.">Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.</a> Nat Protoc. 8 (10), 1837-1840 (2013).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66 (17), 8878-8886 (2006).</li></ol>

在当前的协议中, 我们描述了一个有效的方法单细胞 TCR 放大和随后的 sub-cloning 配对 TCR α和β链成模板逆转录病毒表达载体。虽然, 已经开发了几个单细胞 PCR 协议, 到目前为止没有与直接 sub-cloning 兼容到表达载体。在大多数情况下, 包含高度可变的 CDR3 区域的部分序列被放大, 具有足够的序列来推断变量区域。这种较小的 pcr 产品尺寸支持更高的 pcr 效率, 但需要de 从头TCR 基因合成功能研究?...

t 细胞受体 (TCR) 指示 t 细胞命运决定在发展期间, 稳态/稳态, 和抗原刺激的外围1,2,3。最近的深度测序技术的扩展揭示了先前未被低估的抗原特异 T 细胞反应中的 TCR 多样性。TCR 多样性表明了功能广泛的 T 细胞反应的潜力。为了将 TCR 序列剧目分析与 TCR 功能分析相结合, 测序方法应设计为与实验系统兼容, 并用于后续的功能性研究的体内模型。TCRs我们已经开发了一个有效的方法, 人类 TCR 序列隔离和简化 sub-cloning 成嵌合体的人/小鼠 TCR 模板载体与 TCR 表达的 HLA-人性化的老鼠4。heterodimeric TCRs 的相应α和β链的分离需要从单个细胞中扩增两个链。虽然, 已经开发和利用了几个单细胞的 TCR 克隆协议, 但迄今为止, 没有一个能够很容易地与高通量简化的 Vα/Vβ TCRs 的直接克隆相兼容, 成为重新体内的反转录病毒载体 4、5、6、7。以前的研究采用了两种主要方法, 要么是有选择地放大 TCR 的有限部分足以推断序列, 要么放大整个 TCR 序列4,5,6,7. 通过第一种方法获得的 TCR 序列的下游功能分析需要昂贵的在硅片中程序集和de 从头构造 TCR。虽然第二种方法提供完整的 TCR 序列, 但人的恒定区域与在鼠标模型中克隆的 TCRs 的在体内表达不兼容。我们的方法是专门设计为与在体内的功能分析 TCRs 在小鼠模型中兼容。我们开发了一个高效和简化的单细胞 pcr 协议, 允许直接 sub-cloning pcr 片段到模板表达载体。
我们的方法采用了一个高度敏感的多重嵌套 PCR 反应, 这是两个步骤执行。在第一个复用反应步骤中, 所有 v-β链的40引物的池, 以及所有 v 型α链专用的44个引物池, 用于放大 TCR-α或 TCR-beta, 而无需事先了解序列 (表 1)。前引物有一个适配器序列, 这是纳入 PCR 产品的 5 "。反向底漆基于 TCR 的恒定区域。我们已经确定了在人类变量或连接 TCR 区域中不存在的唯一限制站点, 并将它们合并到新重新设计的 TCRα和 TCRβ引物 (图 1) 中。在第二个嵌套反应中, 使用在恒定区域内的适配器序列和嵌套反向底漆的底漆, 用于进一步放大 TCR 链, 增加特异性 (表 1,图 1)。单细胞分离后, 两轮 pcr (第一反应与池的 Vα和 Vβ引物, 第二次与适配器引物) 产生的 pcr 产品, 可以直接 sub-cloned 到模板逆转录病毒载体。最后的 TCR 结构将编码, 在一个单一的开放阅读框架, 人的可变区域结合的小鼠恒定区域连接的 ' self-cleaving ' 蛋白序列, P2A (hVα-mCα-P2A-hVβ-mCβ)8。P2A 序列已在多个系统中使用, 并已被广泛测试, 专门用于表达 TCRs8、9、10、11。虽然, 在翻译以后多数 P2A 序列仍然附有由一个灵活的链接器连接的阿尔法链子的 C 末端, 而 beta 链信号序列有另外的脯氨酸, 这种修改对 TCR 作用没有有害作用。在构造中使用鼠标常量区域, 而不是人类, 以避免 re-expressed 在鼠标单元格中时与下游信号组件的潜在改变的交互。单一的, 将导致化学计量独立表达α和β链9,11。目前的协议是基于广泛可用的试剂和方法, 并且设计为以流线型和高效的方式进行。虽然我们已经明确地使用这项技术来检测 TCRs 从反应 T 细胞牵连自身免疫性糖尿病, 我们预计, 该协议可以广泛适用于识别和功能评估的人 TCRs 具体自身免疫表位, 肿瘤抗原表位, 或对病原体和疫苗的反应。

<table border="0" cellpadding="0" cellspacing="0" width="815">
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		<tr height="21">
			<td height="21" style="height:21px;width:328px;">CFSE&#160;</td>
			<td style="width:148px;">eBioscience&#160;</td>
			<td style="width:116px;">65-0850-84</td>
			<td style="width:223px;">5 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human CD4 (OKT4) BV421</td>
			<td>Biolegend&#160;</td>
			<td>317433</td>
			<td>2 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human CD3 (OKT3) PerCP/Cy5.5</td>
			<td>Biolegend&#160;</td>
			<td>317336</td>
			<td>2 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse CD3 AF647</td>
			<td>Biolegend&#160;</td>
			<td>100322</td>
			<td>2.5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant RNAse inhibitor biotech grd 2.5KU&#160;&#160;</td>
			<td>VWR</td>
			<td>97068-158</td>
			<td>0.7 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Applied Biosystems High capaity cDNA RT kit&#160;</td>
			<td>Invirtogen&#160;</td>
			<td>4368814</td>
			<td>9 U (RT enzyme)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NF-H2O: Nuclease-free water 1000mL</td>
			<td>Invirtogen&#160;</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gotaq DNA polymerase 2500U</td>
			<td>VWR</td>
			<td>PAM3008</td>
			<td>1 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Half Skirt PCR Plate</td>
			<td>Phenix</td>
			<td>MPX-96M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroAmp Clear Adhesive Film&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>4306311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Agarose&#160;</td>
			<td>Invirtogen&#160;</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SnaBI</td>
			<td>New England Biolabs&#160;</td>
			<td>R0130</td>
			<td>15 U (insert) 30 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SacII</td>
			<td>New England Biolabs&#160;</td>
			<td>R0157</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MfeI</td>
			<td>New England Biolabs&#160;</td>
			<td>R3589S</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BstBI</td>
			<td>New England Biolabs&#160;</td>
			<td>R0519</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calf Intestinal Phosphatase (CIP)</td>
			<td>New England Biolabs&#160;</td>
			<td>M0290S</td>
			<td>10 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quick ligase&#160;</td>
			<td>New England Biolabs&#160;</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Plus minipreps DNA purification systems&#160;</td>
			<td>Promega&#160;</td>
			<td>A1460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Plus midipreps DNA purification systems&#160;</td>
			<td>Promega&#160;</td>
			<td>A7640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA clean &#38; concentrator-25 kit</td>
			<td>Genesee Scientific&#160;</td>
			<td>11-304C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZR-96 DNA clean &#38; concentrator-5 kit</td>
			<td>Genesee Scientific&#160;</td>
			<td>11-306A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subcloning Efficiency DH5a Competent Cells&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>18265017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transit LT-1 Reagent 1mL (transfection reagent)</td>
			<td>Mirus&#160;</td>
			<td>MIR2300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Aria II (sorter)</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Fortessa (flow cytometer)</td>
			<td>BD&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs

虽然, 有几种方法的排序的配对 t 细胞受体 (TCR) α和β链从单一 t 细胞已经开发, 迄今没有有利于下游体内功能分析的 TCR heterodimers。我们已经开发了一个改进的协议的基础上 two-step 多重嵌套 pcr, 这导致一个 pcr 产品横跨整个可变区域的人类 TCR α和β链。通过确定独特的限制点, 并将它们纳入 pcr 引物, 我们已经使 pcr 产品与直接 sub-cloning 与模板逆转录病毒载体相兼容。由此产生的逆转录病毒构建编码一个嵌合的人/鼠 TCR 与鼠标的细胞内域, 这是功能在鼠标细胞或在体内鼠标模型。总的来说, 这里描述的协议结合了人类单细胞配对 TCR α和β链识别与简化的逆转录病毒载体易于适应体外和体内TCR 表达。视频和伴随的材料被设计为对单细胞 PCR 的高度详细的描述, 因此关键的步可以被跟随和潜在的陷阱避免。此外, 我们还提供了生成表达载体所需的克隆步骤的详细描述。一旦掌握, 从单细胞排序到 TCR 表达的整个过程可以在短短的两周内完成。

在步骤 3.2.7 (图 2) 中, 通过在琼脂糖凝胶上5µL 第二反应的运行, 对多重嵌套 PCR 反应的效率进行了检查。平均 TCR-β放大率的效率预计在80% 左右, 而 TCR-α反应的效率通常较低, 约 50%4。只有配对的 TCR α和 TCR β链可以用于 TCR 表达;然而, 所有 PCR 产品都可以测序, 以获得 TCR 序列和剧目信息。
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Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

当前的协议结合了单细胞配对人类 TCR α和β链测序与与体外和体内TCR 表达相兼容的反转录病毒载体的生成。

简化的单细胞 TCR 分离和逆转录病毒载体的生成, 用于体外和体内表达人类 TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Research

JoVE Journal

Immunology and Infection

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...