Name: isolation, transfection, and culture of primary human monocytes
Uploaded: 2019-12-16T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

作者要感谢HIV临床/肿瘤生物储存核心提供患者样本和细胞免疫学代谢核心提供流式细胞测定分析。该项目由NIH P20GM121288和P30GM114732资助。

下面描述的所有方法都已获得路易斯安那州立大学健康科学中心新奥尔良机构审查委员会的批准。所有血液都是在征得知情同意后采集的。
注：整个过程在生物安全2级（BSL2）设施中的无菌条件下进行，以便谨慎处理生物材料。特别是，每个步骤都是在生物安全柜下使用无菌技术执行的。在涉及血液、血液制品、细胞或细胞产品移液的每一步后，在正确处置之前，使用罩内废...

<ol>
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提出的方案演示了使用HIV感染者的初级细胞作为研究单核细胞和巨噬细胞的模型。HIV–接受 CART 的患者感染了多年，并且可能还有其他与免疫系统受损相关的共同感染。为了研究在HIV慢性感染的情况下的免疫调节，细胞直接从患者身上采集。由于miRNA已被证明在细胞发育和分化中起着重要作用，该协议侧重于在这些主细胞中操作miRNA表达的能力（图2，...

人体免疫缺陷病毒-1（HIV-1）感染导致严重的免疫功能障碍，可导致机会性感染和后天免疫机能丧失综合症（艾滋病）。虽然接受cART的HIV感染者的特点是病毒载量低和正常的CD4+T细胞计数，免疫系统的功能可能在这些领域受损，导致免疫反应功能失调，这与患癌症的风险增加有关。CART的HIV患者的免疫功能障碍机制在很大程度上仍不为人所知。因此，描述患者衍生的免疫细胞并研究其生物学和功能是当前艾滋病毒研究的重要组成部分。
单核细胞和巨噬细胞是免疫反应的主要调节者，在HIV感染2、3、4、5中起着基础性作用。异构和塑料在自然界中，巨噬细胞可以大致分为经典激活（M1）或替代激活（M2）。虽然这种一般分类在设置实验条件时是必要的，但巨噬细胞的极化状态可能被各种细胞因子6、7、8、9逆转。虽然有多项研究已研究爱滋病毒感染对单核细胞和树突细胞的影响，但单核细胞介导反应的分子细节基本上未知6、7、10、11、12、13、14、15、16、17、18、19。在免疫细胞调节和功能所涉及的因素中，微RNA（miRNA），即转录后调节基因表达的短非编码RNA，已被证明在主要细胞通路（即生长、分化、发育和凋亡）20中发挥重要作用。这些分子被描述为转录因子的重要调节器，对控制巨噬细胞21的功能极化至关重要。miRNA在接受CART的HIV感染者的单核细胞中的潜在作用已经调查，但该领域的进展需要更多的工作22，23，24，25，26。本文讨论了一种从HIV感染者和对照组将miRNA和siRNA转染成原发人类单核细胞的优化方法。
该协议依赖于市售试剂和试剂盒，因为技术程序的连续性有助于消除临床样本工作时不必要的实验变量。尽管如此，该方法提供了重要的提示（即，用无血清培养基培养的细胞数量或短暂孵育，以促进细胞粘附在板中）。此外，该协议中使用的极化条件派生自已发表的作品27、28、29。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K2EDTA</td>
			<td>BD Biosciences</td>
			<td>366643</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Stain Buffer</td>
			<td>BD Horizon</td>
			<td>563794</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD14 PerCP</td>
			<td>Invitrogen</td>
			<td>46-0149-42</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD163 BV711</td>
			<td>BD Horizon</td>
			<td>563889</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD209 BV421</td>
			<td>BD Horizon</td>
			<td>564127</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD80 FITC</td>
			<td>BD Horizon</td>
			<td>557226</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD83 APC</td>
			<td>BD Horizon</td>
			<td>551073</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Sep Direct Human Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19669</td>
			<td></td>
		</tr>
		<tr>
			<td>EIF4EBP1 mAb</td>
			<td>Cell Signaling</td>
			<td>9644</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>EIF4EBP1 siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-29594</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovin Serum Defined Heat Inactivated</td>
			<td>Hyclone</td>
			<td>SH30070.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallios Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>B43618</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH mAb</td>
			<td>Santa Cruz</td>
			<td>SC-47724</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>HuFcR Binding Inhibitor</td>
			<td>eBiosciences</td>
			<td>14-9161-73</td>
			<td>Flow cytometry- blocking buffer</td>
		</tr>
		<tr>
			<td>Kaluza Analysis Software</td>
			<td>Beckman Coulter</td>
			<td>B16406</td>
			<td>Software to analyze flow cytometry data</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O55:B5</td>
			<td>Sigma</td>
			<td>L4524</td>
			<td></td>
		</tr>
		<tr>
			<td>miRCURY LNA microRNA Mimic hsa-miR-146a-5p</td>
			<td>Qiagen</td>
			<td>YM00472124</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION miRNA Negative Control</td>
			<td>Sigma</td>
			<td>HMC0002</td>
			<td>Scrambled miRNA conjugated with a near infrared dye</td>
		</tr>
		<tr>
			<td>Nunc 35mm Cell Culture Dish</td>
			<td>Thermo Scientific</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human GM-CSF</td>
			<td>R&#38;D Systems</td>
			<td>215-GM-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>R&#38;D Systems</td>
			<td>204-IL-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>R&#38;D Systems</td>
			<td>216-MC-025</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-Glutamine</td>
			<td>Corning</td>
			<td>10040CVMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Scrambled Control siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-37007</td>
			<td></td>
		</tr>
		<tr>
			<td>Viromer Blue Transfection Reagent Kit</td>
			<td>Lipocalyx</td>
			<td>VB-01LB-01</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-1 Cell Proliferation Reagent</td>
			<td>Roche</td>
			<td>5015944001</td>
			<td>Colorimetric assay to assess cell viability</td>
		</tr>
	</tbody>
</table>

isolation, transfection, and culture of primary human monocytes

尽管1990年代中期采用了联合抗逆转录病毒疗法（cART），但人体免疫机能丧失病毒（艾滋病毒）仍然是一个重大的健康问题。虽然抗逆转录病毒疗法能有效地降低全身病毒载量并恢复正常的CD4+ T细胞计数，但它并不能重建一个完全功能的免疫系统。接受CART的HIV感染者免疫系统功能失调，其特征可能是免疫激活、免疫细胞早衰或持续炎症。这些情况，以及与艾滋病毒感染有关的合并因子，增加了疾病的复杂性，在细胞和动物模型中不容易重现。为了研究这些患者免疫功能障碍背后的分子事件，这里提出了一个在体外培养和操纵人类原发性单核细胞的系统。具体来说，该议定书允许从接受CART的艾滋病毒感染者以及艾滋病毒阴性对照中获得的原发性CD14和单核细胞的培养和转染。该方法涉及单核细胞和单核衍生巨噬细胞的分离、培养和转染。虽然使用了市售试剂盒和试剂，但该协议为使用miRNA模拟和抑制剂以及siRNA成功粘附和转染单核细胞提供了重要提示和优化条件。

使用所述程序，从艾滋病毒感染者和健康捐赠者中分离出主要的人类单核细胞。这里提供的所有数据均来自艾滋病毒和接受低（<20份/mL）或检测不到的病毒载量和正常CD4+T细胞计数的CART的受试者。分离后，细胞立即被染色，并进行流动细胞测量，以确认细胞群的纯度。结果显示，超过97%的细胞对CD14的染色呈阳性（数据未显示）。对于巨噬细胞的极化，使?...

Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

Isolation, Transfection, and Culture of Primary Human Monocytes

这里介绍的是一个优化的协议，用于分离、培养、转染和区分人类原发性单核细胞与艾滋病毒感染者和健康对照。

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. ...

HIV 感染导致免疫功能障碍，即使在接受抗逆转录病毒治疗的个人中，也未发现病毒。这种方法允许研究单细胞功能，这是免疫反应的关键调节器。我们优化了这种技术，用于人类原发单核细胞的分离、培养和转染。我们使用这种方法来了解HIV感染者免疫功能障碍的分子机制，但它可以应用于涉及单核细胞的任何其他免疫研究。如果这是你第一次使用人类血液，请记住遵循正确的生物安全协议，并处理所有样品作为可能感染性物质。在四个10毫升EDTA真空管中收集40毫升新鲜人类全血后，使用无菌技术将所有血液转移到生物安全柜中的单个50毫升锥形丙烯管中。按照制造商从选定的人类单细胞分离试剂盒的指示，将两毫升单核细胞分离鸡尾酒从试剂盒添加到血管中，然后从试剂盒中旋转磁珠 30 秒。在血液中加入两毫升珠子，并使用 25 毫升血清移液器小心地将珠子与血液混合。在室温下五分钟后，将血液平均分割在四个50毫升管之间，并添加30毫升无菌PBS，并辅以一毫摩尔EDTA。再次与塑料 25 毫升血清移液器混合，将管子放在磁性支架中。10分钟后，使用移液器从每个管的中心绘制内容物，注意不要吸气超过一毫升红血球，并分配管内物到四个新的50毫升管之一。将涡旋磁珠的500微升加入每个管中，轻轻混合细胞溶液。将管子放回磁架5分钟，然后小心地将内装物从每个管的中心转移到四个新的50毫升管中。当所有细胞悬浮液都转移后，将每个新的50毫升管放入磁体支架5分钟，然后小心地将管内板转移到第三组四个新的50毫升管中。然后通过离心收集细胞，并在总共10毫升无菌PBS中重新供应所有四个细胞颗粒进行计数。计数后，将分离的单核细胞以每毫升10倍至6个细胞的37摄氏度无血清RPMI 1640介质补充抗生素，将一毫升再增的细胞重新放入生物安全柜中的35毫米板中。然后将板放在细胞培养箱中30至60分钟，使细胞粘附在井底。为了使用M1样表型的巨噬细胞，在每个盘子里加入100微升的胎儿牛血清，每毫升GM-CSF含有25毫微克。为了使用M2样表型的巨噬细胞，向每个板中加入100微升的胎儿牛，每毫升M-CSF含有50毫微克。对于具有微RNA模拟、抑制剂或小干扰RNA的单细胞转染，按照制造商关于转染试剂盒的协议，将缓冲液中选定的RNA稀释至10微升的最终浓度为1.83微摩尔，其中10微升稀释的模拟或抑制剂的转染量为10倍至第五细胞体积。要准备转染试剂，在1.5毫升微离心管中加入一微升聚合物，并立即加入90微升试剂盒提供的缓冲液，每转染获得91微升试剂。涡流摄政3至5秒，移液器90微升的转染溶液进入管内含有10微升稀释RNA。轻轻混合后，在室温下孵育溶液15分钟，然后向一个细胞井中加入100微升的转染复合物。在37摄氏度下4小时后，用含有适当生长因子的三毫升完全介质代替该介质。在培养的第六天，用三毫升的培养剂代替来自M1培养物的培养剂，补充胎儿牛血清、抗生素、脂质多糖和干扰素伽马，用中辅胎儿牛血清、抗生素、M-CSF和白细胞-4取代M2培养物的超级天然剂。培养24小时后，每次洗涤用新鲜PBS洗涤每盘活性巨噬细胞两次。对于RNA和蛋白质分析，将附着的细胞直接在板中，以便收集释放的细胞内容进行分析。对于流式细胞测量分析，在37摄氏度下用PBS中两毫摩尔EDTA分离细胞10分钟，然后轻轻地从板底刮取细胞，以便将其收集到1.5毫升微离心管中进行分析。这里显示了M1-激活对照组和患者衍生细胞的代表性直方图，与非激活的T0细胞相比，CD80和CD83和M2激活细胞水平增加。有趣的是，CD80和CD83在控制源细胞中表达比HIV衍生细胞更强烈。新鲜收集的单核细胞的转染与炒近红外标记的微RNA产生超过90%的转染效率，由流式细胞学和共物显微镜确定。此外，无论细胞成熟阶段或转染条件如何，转染不会显著降低患者衍生细胞或对照细胞的生存能力。转染导致在分离后的第一天和第四天，通过特定的小干扰RNA对转染时的目标基因进行有效的降低调节。此外，通过微RNA模拟转染的细胞表明，与未转染的细胞的微RNA表达增加48至72倍，而所有转染对等控制都无明显变化。镀的细胞数量对生存至关重要。我们每35毫米培养皿推荐100万个细胞。在无血清介质中电镀也会大大改善细胞附着。使用这种方法获得的细胞可以使用细胞因子或生长因子进行治疗，以研究患者与健康对等患者的不同反应。

Research

JoVE Journal

Immunology and Infection

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

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 ...

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 ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

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. ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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

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 ...

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 ...