This work was supported by the US National Institutes of Health grants (R01HL109102, P01HL107202, U19CA179512, F31HL131361), a Leukemia &amp; Lymphoma Society scholar award (K.M.A.), and the National Institute of General Medical Sciences (NIGMS) Medical Scientist Training Program (Grant #T32GM007618) (M.M.).

该协议坚持加州大学旧金山分校的人类研究伦理指导原则。 1. T细胞培养，CD4 + T细胞的分离，和Th17极化的制备<ol><li> 在第0天，涂层6孔，每个抗人CD3（2微克/ mL）和抗人CD28的孔1.5ml组织培养板（4微克/毫升）的PBS与钙和镁为至少2小时37℃。 <ol><li>可替代地，涂层在4℃下过夜平板。裹在板封口膜。 </li></ol></li><li>制备脐带血单核细胞（CBMCs）通过根据制造商说明书密度梯度离心。 注意:仔细工作，确保适当的个人防护装备（PPE）处理人血的时候以避免暴露于血源性病原体的风险已磨损。 </li><li> 一旦单核细胞已被分离和洗涤，通过负塞莱执行人CD4 + T细胞分离ction使用市售的人CD4 + T细胞的试剂盒。 注意:如果有后单核细胞的分离红血细胞污染，任选的红血细胞裂解可以先于CD4 + T细胞分离的步骤中进行。重悬的单核细胞在1mL隔离缓冲液（2％FBS的PBS中）的。加入5毫升的1X溶胞液。孵育在室温下15分钟。然后在300×g下在4℃下加入5毫升分离缓冲液和离心机5分钟以沉淀细胞。 <ol><li>重悬的单核细胞在隔离缓冲器一个的50×10 6密度为每500μL在一个新的5mL的试管中。 </li><li>加入100μLFBS和100μL抗体混合物每管，然后在4℃下孵育各管20分钟定轨振荡器上充分混合的。 </li><li>在温育后，加入隔离缓冲器3-4 mL至洗涤细胞。离心将细胞在300×g离心8分钟，在4℃以沉淀细胞。小心吸苏佩rnatant。 </li><li>在这个旋转，预洗磁珠。转移的磁珠的所需量进新管中（在1中使用:1与细胞），并添加隔离缓冲器的等体积的洗涤珠。拌匀使用1种毫升微量，然后将管在磁体至少1分钟。小心吸出上清液。重悬在相同体积的初始前的洗涤传送的磁珠。 </li><li>将细胞重悬于每个管与隔离缓冲器500μL，并添加预洗涤磁珠500μL。 </li><li>孵育在室温下（18℃至25℃）的定轨摇床上以15分钟的磁珠的细胞拌匀。 </li><li>孵育后，彻底吸管使用1毫升微量的至少10倍的细胞。然后添加隔离缓冲器的3-4毫升，并放置各管在磁体2分钟。 </li><li>小心转移是在带负选择的CD4 + T细胞上清至新管。 </li></ol></li><li>一旦CD4 + T细胞分离完成后，计数与血球细胞和保持细胞在冰上。 </li><li>用PBS洗涤抗体包被的平板两次。然后1.5 Th17的偏振媒体的2倍的溶液混合添加到每个孔中:抗人IFNγ（20微克/毫升），抗人IL-4（20μg/ mL的），人TGFβ（10ng / mL的），人IL-1β（40毫微克/毫升），人IL-23（40毫微克/毫升），人IL-6（50毫微克/毫升）的所有稀释在无血清基础培养基（补充有2mM L-谷氨酰胺，100 U / ml的青霉素，100μg/ mL链霉素，10mM的HEPES，1mM丙酮酸钠和100μM2-巯基乙醇）。 注:转染和RNA干扰确实在含血清媒体工作。使用无血清培养基与该协议的目的是为了实现更好的IL-17A的生产。 </li><li> 离心机人类CD4 + T细胞在500×g离心5分钟，在4℃以沉淀细胞。小心吸supernat蚂蚁。然后重悬在无血清基础培养基的细胞和板上的细胞以3×10 6个的密度在每孔，使得最终体积为每孔3 mL和偏振细胞因子现在正处于1X终浓度1.5毫升。 <ol><li>将平板在5％CO 2，37℃的培养箱中两天。 </li></ol></li></ol> 2. 体外的电穿孔极化人Th17细胞<ol><li> 第2天，外套48孔每抗人CD3（2微克/ mL）和抗人CD28的井250μL的组织培养板（4微克/毫升）的PBS与钙和镁为至少2小时37℃。 <ol><li>可替代地，涂层在4℃下过夜平板。裹在板封口膜。 </li></ol></li><li>准备小RNA转染。对于每次转染，等分试样1 5μM储备溶液的siRNA到1.5mL微量离心管中的μL。包括适当的化学匹配的小RNA对照醇。置于冰上所有的管子。 </li><li>用PBS洗涤抗体包被的平板两次。然后添加500μL的1X的Th17极化介质到每个孔中:抗人IFNγ（10微克/毫升），抗人IL-4（10μg/ mL的），人TGFβ（5毫微克/毫升），人IL- 1β（20ng / mL的），人IL-23（20ng / mL的），人IL-6（25毫微克/毫升）的所有稀释在无血清基础培养基（补充有2mM L-谷氨酰胺，100U / mL青霉素，100微克/毫升链霉素，10mM的HEPES，1mM丙酮酸钠和100μM2-巯基乙醇）。 </li><li>制备的培养板中并转染试剂后，重悬以1种毫升微量，轻轻移液，但要确保所有的细胞从孔的底部分离的细胞。池中的细胞进入一个锥形管和离心机在500×g下在4℃下5分钟。 注:对于除本文所提出的四天协议更长的培养期间，细胞应大致使用相同的协议转染的每3天。第2步0.4应当但是修改，因为用不同的小RNA处理的细胞不应该被合并。所有的被测试的条件必须收集，计数，并单独转染。 </li><li>小心吸出上清液，然后重悬细胞中至少1毫升的PBS洗涤。计数活Th17细胞（ 例如 ，使用台盼蓝排除来评估活力血球），然后将细胞转移到一个离心管中。 </li><li>离心在500xg 5分钟在室温下使细胞沉淀。 </li><li>小心吸出上清液，然后使用从转染系统10μL试剂盒所提供的重悬缓冲液以每毫升2.5-4×10 7个细胞的密度悬浮细胞。保持细胞在室温下。 注:作为指导原则，尽量只准备尽可能多的细胞可以在30分钟内被转染。多批次可以比拟的。 </li><li>添加细胞的9μL到的小RNA 1μL在每个的MicroCentrifuge管。移液管一次混合用于转染细胞和小RNA然后加载到移液管提供电极尖端。 注意:通常情况下，添加细胞悬浮液的9.5μL，以确保有足够的混合物，以防止在转染前吸管电极末端产生气泡。 </li><li>用3mL室温电解缓冲液E.地点的吸液管站内的反应杯填充提供反应杯，然后将吸管到试管内的位置。 </li><li>立即电穿孔使用下列参数细胞和小RNA的各10μL混合物:脉冲电压1,500-1,550伏，脉冲宽度为10ms，和3个脉冲总转染装置上。 </li><li>电穿孔完成后，直接在细胞混合物添加到1X的Th17偏振媒体在一个培养板的孔中制备的500μL。代替板在5％CO 2，37℃的培养箱中两天。 注意:通过移液洗净移液管尖电极起来，并在PBS中向下每次转染之间。 </li></ol> 3.收获人Th17细胞<ol><li>重悬在培养基中的细胞微量，轻轻吹打，但要确保所有的细胞是从孔的底部分离。准备功能和/或基因表达分析的细胞。 注意:常规地，足够的细胞被转染的Th17细胞的单个孔中，得到用于表面标记物和细胞内细胞因子和转录因子染色的流式细胞术分析或用于制备RNA的用于基因表达分析。 </li></ol>

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The authors have nothing to disclose.

该协议提供用于递送的小RNA的成人Th17细胞的改进的方法。虽然人Th17细胞在此使用，电穿孔的这种方法与小RNA可以与其他主人T辅助细胞亚组，如TH1，TH2，以及调节性T细胞可以使用。它不是幼稚的CD4 + T细胞的运作良好，因此细胞必须培养的转染之前被激活。对于此协议中，我们首先优化了体外培养体系为更好的IL-17A的生产。最大的因素是基础的媒体。我们发现，无血清培养基优于传...

CD4 + T细胞是适应性免疫应答的关键协调器。幼稚CD4 + T细胞是能够发育成几个不同的效应T细胞（ 例如 TH1，TH2，Th17细胞， 等 ），每个具有自己的一套特性的细胞因子和转录因子，取决于局部微环境1。血统决定T细胞使对于两个保护性免疫和容忍，自我批评。 Th17细胞是公知的，以打击胞外细菌和真菌的T细胞的一个子集，但它们的不当响应也牵涉在多种自身免疫疾病和炎性疾病的发病机理，例如多发性硬化和牛皮癣2,3。人Th17细胞可以从幼稚的CD4 + T细胞在体外通过提供一个适当的偏振环境4来生成。 VARIO<html>我们的细胞因子的组合IL-1β，IL-23，TGFβ，和IL-6已被用于人Th17细胞的发展。人Th17细胞表达CCR6，即通常用于识别该细胞群，并通过它们的主要转录因子，RORγt（由RORC编码）5,6的表达被定义趋化因子受体。 Th17细胞具有表达多种细胞因子的能力，但IL-17A是由这些细胞产生的细胞谱系限定的效应细胞因子。我们检查了所有三个Th17细胞相关标记（CCR6，RORγt，IL-17A）的表达来评估我们的人Th17 体外分化检测的鲁棒性。此外，我们培养非偏振的条件下，在没有细胞因子或阻断抗体添加到培养基中作为阴性对照使用，因为这些Th17细胞标志物的表达应该是非常低的或不存在下的人CD4 + T细胞。 ve_content"&gt;学习正常的人T细胞的发育和生物学的一种方法是其发展过程中操纵基因表达。短干扰RNA（siRNA）是靶向蛋白质编码的mRNA，并且可以被利用来降低特定基因表达的合成的小RNA分子。微RNA（miRNA）是公知的调节基因表达的转录后的内源性非编码小RNA。的miRNA已显示出在小鼠和人的T细胞生物学中起重要作用，包括在Th17细胞7，8，9，它关键的是要具有在人T细胞操纵的小RNA的活性，研究其对基因表达，并最终对人T细胞生物学效应的可靠的方法。在此，我们描述了我们用于引入开发了一种易于使用的，一致的和可靠的协议小合成的RNA和锁定核酸（LNA，化学修饰的核酸具有增加的稳定性）成免疫细胞，并且具体到人Th17细胞。 有引入的小RNA到哺乳动物细胞中，其通常落入化学，生物或物理类别10的若干替代方法。常用的化学方法，包括基于脂质的转染和磷酸钙转染，依靠创建被细胞更有效地吸收化学-DNA复合物。一般而言，化学方法是不适合的原代T细胞的转染效率高。最常见的生物的方法是使用病毒载体（ 例如逆转录病毒或慢病毒），其直接插入外来RNA引入宿主作为其天然复制周期的一部分。病毒转导通常需要更长的时间来完成，特别是当一个在时间因素的原病毒的质粒分子克隆。此外，病毒转导载体可以是可能危害人体研究。电穿孔是一种物理米通过使细胞对高的电压脉冲，从而允许核酸以瞬时进入细胞在那里他们可以在他们的目标作用诱导膜通透性ethod。传统的电文书不能有效转染主要淋巴细胞。然而，优化的下一代电穿孔已经被证明是能够以很高的效率转染的T细胞，特别是当待转染的材料为小RNA。术语下一代是松散用来区分传统的电穿孔机两个新的平台（ 例如 ，霓虹灯，Amaxa公司）。此外，此方法是一种用于具有高达在单一实验中约120的小RNA，其通常使用验证合成试剂中等通量筛选容易扩展。重要的是，转染的成功可以在T细胞活化后只需16小时来实现。这种方法的缺点，但是，它不会导致基因组稳定incorporati上，因此是短暂的。因此，这是值得的额外的努力以创建可以被包装到病毒载体中并在T细胞在需要小RNA的长期表达的情况下成功地表达了稳定的表达构建体。 我们已经使用了下一代的转染（ 例如 ，氖），实现多样化合成的单链或双链RNA或LNA寡核苷酸的工具用于不同的目的11，12，13。有效的RNA干扰可以在原代小鼠和人T细胞使用双链短干扰RNA（siRNA）所诱导。这个协议描述了用于在人Th17细胞使用这种技术的优化条件。除了的siRNA，可商购的合成的miRNA模拟物和抑制剂可用于研究的miRNA增益和功能丧失。 miRNA模拟物是双链RNA分子非常相似的siRNA，但designe20d与内源成熟miRNA的序列。 miRNA的抑制剂是经化学修饰的结合天然miRNA和拮抗它们的功能的RNA和/或LNA基于单链寡核苷酸。我们发现，所有这些工具都可以在原代培养的T淋巴细胞有效地使用，包括但不限于人Th17细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-human IL-17A PE</td>
			<td>ebioscience</td>
			<td>12-7179-42&#160;</td>
			<td>Clone: eBio64DEC17</td>
		</tr>
		<tr>
			<td>anti-human IFNg FITC</td>
			<td>ebioscience</td>
			<td>11-7319-82</td>
			<td>Clone: 4S.B3</td>
		</tr>
		<tr>
			<td>anti-human CD4 eVolve605</td>
			<td>ebioscience</td>
			<td>83-0047-42</td>
			<td>Clone: SK3</td>
		</tr>
		<tr>
			<td>mouse anti-human CD196 (CCR6) BV421</td>
			<td>BD biosciences</td>
			<td>562515</td>
			<td>Clone: 11A9</td>
		</tr>
		<tr>
			<td>anti-human RORgt AF647</td>
			<td>BD biosciences</td>
			<td>563620</td>
			<td>Clone: Q21-559</td>
		</tr>
		<tr>
			<td>anti-human CD45 eFluor450</td>
			<td>ebioscience</td>
			<td>48-9459-42</td>
			<td>Clone: 2D1</td>
		</tr>
		<tr>
			<td>Foxp3/Trascription Factor Staining Buffer Set</td>
			<td>ebioscience</td>
			<td>00-5523-00</td>
			<td>For intracellular transcription factor flow cytometry staining</td>
		</tr>
		<tr>
			<td>Hu FcR Binding Inhibitor Purified</td>
			<td>ebioscience</td>
			<td>14-9161-71</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoCult-XF T Cell Expansion Medium</td>
			<td>Stemcell Technologies</td>
			<td>10981</td>
			<td>&#34;Serum-Free Base Media&#34;</td>
		</tr>
		<tr>
			<td>MACS CD28 pure functional grade, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-375</td>
			<td>Clone: 15E8</td>
		</tr>
		<tr>
			<td>anti-human CD3 Purified</td>
			<td>UCSF monoclonal antibody core</td>
			<td>N/A</td>
			<td>Clone: OKT-3</td>
		</tr>
		<tr>
			<td>LEAF purified anti-human IL-4</td>
			<td>Biolegend</td>
			<td>500815</td>
			<td>Clone: MP4-25D2</td>
		</tr>
		<tr>
			<td>anti-human IFNg, functional grade purified</td>
			<td>ebioscience</td>
			<td>16-7318-85</td>
			<td>Clone: NIB42</td>
		</tr>
		<tr>
			<td>Recombinant human IL-23</td>
			<td>Peprotech&#160;</td>
			<td>200-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-1&#946;</td>
			<td>Peprotech&#160;</td>
			<td>200-01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TGF-&#946;1</td>
			<td>Peprotech&#160;</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-6</td>
			<td>Peprotech&#160;</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>siGENOME Control Pool, Non-targeting #2</td>
			<td>Dharmacon</td>
			<td>D-001206-14-05</td>
			<td></td>
		</tr>
		<tr>
			<td>siGENOME SMARTpool Human RORC</td>
			<td>Dharmacon</td>
			<td>M-003442-00</td>
			<td></td>
		</tr>
		<tr>
			<td>siGENOME SMARTpool Human PTPRC</td>
			<td>Dharmacon</td>
			<td>M-008067-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Untouched Human CD4 T Cells Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>11346D</td>
			<td>Human CD4+ T cell Isolation Kit</td>
		</tr>
		<tr>
			<td>Neon Tranfection System</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td>Next generation electroporation instrument</td>
		</tr>
		<tr>
			<td>Neon Tranfection System 10 &#956;l Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Resuspension Buffer T</td>
			<td>ThermoFisher Scientific</td>
			<td>Provided in kit (MPK1096)</td>
			<td>&#34;Transfection Resuspension Buffer&#34;</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>#07801</td>
			<td>Density Gradient Medium</td>
		</tr>
		<tr>
			<td>costar 6-well tissue culture treated plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>flat bottom plates</td>
		</tr>
		<tr>
			<td>costar 48-well tissue culture treated plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>flat bottom plates</td>
		</tr>
		<tr>
			<td>BD Pharm Lyse lysing buffer, 10 X</td>
			<td>BD biosciences</td>
			<td>555899</td>
			<td>Must make 1X solution with distilled water prior to use</td>
		</tr>
	</tbody>
</table>

small rna transfection in primary human th17 cells by next generation electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

第一步，显影的成功电穿孔人Th17细胞的可靠系统是产生在体外分化的人Th17细胞培养物的鲁棒性。 Th17的偏振条件下培养的T细胞中表达的趋化因子受体CCR6和转录因子RORγt（ 图1A，左）。当T细胞非偏振（THN）的条件下（ 图1A，右图）培养没有表达这些标记物。 Th17的偏振条件下培养的T细胞也取决于再刺激产生的IL-17A（ 图1B，左侧...

Watch this Scientific Journal Video about Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation at JoVE.com

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

small-rna-transfection-primary-human-th17-cells-next-generation

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  University of California, San Francisco. Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

视频: Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

科研

免疫与感染

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

原人类单核细胞的隔离、转染和文化

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...