1。隔离的PBMC <ol><li>绘制成一个BD真空采血管彩管的细胞与肝素钠的制备管血（7.5-8.0毫升）。管含有钠抗凝和聚蔗糖Hypaque密度流体，加上聚酯凝胶屏障，这两种液体分开。在一个水平旋翼为20分钟1800 × 克 （摇摆头），在室温（18℃至25℃）离心管/血液样本。切换离心机刹车关闭。 </li><li>离心后，层的顺序发生如下（从上到下看到）：一）等离子体 - B）外周血单个核细胞（PBMC）和血小板...

<ol>
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这里介绍的方法，使γδT细胞从外周血单个核细胞的有效扩张。 γδT细胞激活，并通过zoledronate扩大和IL - 2制定完整的效应功能，细胞因子的产生和细胞毒性反映。据报道，合成phosphoantigens bromohydrin焦（BrHPP）和2 - 甲基3 - 丁烯-1 - 焦磷酸（2M3B1PP）也扩大γδT细胞，但是，他们不市售。相比之下，zoledronate已经授权作为择泰的临床应用。因此，一个可靠的试剂是容易获得。 培养?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>择泰</td><td>诺华制药K. K </td><td></td><td> zoledronate </td></tr><tr><td> PROLEUKIN </td><td>诺华制药</td><td></td><td>重组人IL - 2 </td></tr><tr><td> BD真空采血管彩管细胞制备与肝素钠管</td><td>屋宇署</td><td> 362753 </td><td></td></tr><tr><td>的RPMI1640 </td><td> Invitrogen公司</td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203介质</td><td>细胞科学与技术学院</td><td> 0301-7 </td><td></td></tr><tr><td> OpTmizer </td><td> Invitrogen公司</td><td> 0080022SA </td><td></td></tr><tr><td> brefeldin一个</td><td>西格玛</td><td> B5936 - 200UL </td><td></td></tr><tr><td>佛波醇-12 - 肉豆蔻13醋酸乙烯酯（PMA） </td><td>西格玛</td><td> P1585 - 1MG </td><td></td></tr><tr><td>离子霉素</td><td>西格玛</td><td> 13909 - 1ML </td><td></td></tr><tr><td> IntraPrep </td><td> Beckman Coulter公司</td><td> A07803 </td><td></td></tr><tr><td>抗人CD3 - FITC或PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td>抗人CD4 - ECD </td><td> Beckman Coulter公司</td><td> 6604727 </td><td></td></tr><tr><td>抗人类CD8-PE/Cy5 </td><td> Beckman Coulter公司</td><td> 6607011 </td><td></td></tr><tr><td>抗人类CD14-PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07765 </td><td></td></tr><tr><td>抗人CD19 - PE </td><td> Beckman Coulter公司</td><td> A07769 </td><td></td></tr><tr><td>抗人CD45 - ECD </td><td> Beckman Coulter公司</td><td> A07784 </td><td></td></tr><tr><td>抗人类CD56-PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07789 </td><td></td></tr><tr><td>抗人TCRαβ- PE </td><td> Beckman Coulter公司</td><td> A39499 </td><td></td></tr><tr><td>抗人TCRVγ9- FITC </td><td> Beckman Coulter公司</td><td> IM1463 </td><td></td></tr><tr><td>抗人类CD27-PE/Cy5 </td><td> Beckman Coulter公司</td><td> 6607107 </td><td></td></tr><tr><td>抗人CD45RA - ECD </td><td> Beckman Coulter公司</td><td> IM2711 </td><td></td></tr><tr><td>抗人CD69 - PE </td><td>屋宇署</td><td> 555531 </td><td></td></tr><tr><td>抗人NKG2D的聚乙烯</td><td> Beckman Coulter公司</td><td> A08934 </td><td></td></tr><tr><td>反humalIFNγ的聚乙烯</td><td> Beckman Coulter公司</td><td> IM2717U </td><td></td></tr><tr><td>鼠IgG1同型对照PE </td><td> Beckman Coulter公司</td><td> A07796 </td><td></td></tr><tr><td>鼠IgG1同型对照ECD或PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

人类γδT细胞能够识别和响应到多种应激性抗原，从而开发先天广泛的抗肿瘤和抗感染活性。1外周血中大多数γδT细胞的Vγ9Vδ2T细胞受体。这些细胞识别抗原在主要组织相容性复合体独立的方式，并发展强大的杀伤和Th1样的效应功能。因此，γδT细胞是肿瘤免疫治疗的有吸引力的候选人的效应细胞。 Vγ9Vδ2T细胞响应，如phosphoantigens（E）4 -羟基- 3 -甲基- 2 -己烯焦磷酸（HMBPP），这是通过类异戊二烯生物合成的细菌合成;和异戊烯基焦磷酸（IPP），这是生产在真核细胞通过甲羟戊酸途径，在生理条件下 ，3中，IPP的nontransformed细胞的生成是没有足够的γδ T细胞的活化。甲羟戊酸途径在肿瘤细胞失调， 导致3，因为IPP的积累和γδT细胞的活化。aminobisphosphonates（如帕米膦酸或zoledronate）抑制法尼基焦磷酸合成酶（制剂），酶的作用IPP的甲羟戊酸途径下游，细胞内水平IPP和sensitibityγδT细胞识别，可以治疗由aminobisphosphonates增加。 IPP的积累是低效率的nontransfomred细胞比肿瘤细胞，使我们对癌症与aminobisphosphonatesγδT细胞通过激活免疫药理相关浓度的aminobisphosphonates，4有趣的是，IPP的积累在单核细胞的PBMC与aminobisphosphonates治疗，因为高效，这些细胞的药物摄取。积累的IPP成为抗原提呈细胞和Vγ9Vδ2T细胞刺激外周血单核细胞。基于这些机制，我们制定了一个大规模扩张γδT细胞zoledronate文化和白细胞介素的技术-2（IL - 2）7其他方法扩大γδT细胞利用合成phosphoantigens bromohydrin焦（BrHPP）8或2 -甲基-3 -丁烯- 1 -焦磷酸（2M3B1PP）9，所有这些方法允许前体外扩增，γδT细胞过继免疫治疗中使用的大量产生。然而，只有zoledronate是一个FDA批准的市售试剂。 Zoledronate扩大γδT细胞显示CD27 - CD45RA -效应记忆体的表型和它们的功能可以通过干扰素γ的生产检测7评估。

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  Î³Î´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

扩大γδT细胞从外周血单个核细胞（PBMC）的一个方法来描述。外周血单核细胞衍生的γδT细胞受到刺激和扩大使用zoledronate和白细胞介素2（IL - 2）。 γδT细胞的大规模扩张，可应用于自体肿瘤细胞免疫疗法。

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Research

JoVE Journal

Immunology and Infection

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

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 Human Alloantigen-specific T Cells from Peripheral Blood

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

使用微泡从人外周血单核细胞样品中分离、活化和扩增基于浮选的 T 细胞

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

功能性破骨细胞与人外周血CD14+ 单核细胞的鉴别

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...