这项研究是由美国国立卫生研究院授予K08 AI062732，K12的HD001401，并1UL1RR024139 - 02和儿童健康，从查尔斯H ·胡德基金会研究资助SB - M。在纽约州立大学石溪分校的研究基金会。

1。 EB病毒备料<ol><li>继代培养的急剧增长的一个75厘米2组织培养烧瓶使用无菌技术B95 - 8细胞（ATCC CRL，1612; 13）在3 × 10 5细胞/ ml。细胞是含10％热灭活胎牛血清（FBS），，青霉素/链霉素在100U/ml和100微克/毫升，分别增长完全RPMI 1640和两性霉素B在0.5微克/毫升，37 °的存在彗星5％的CO 2。 </li><li>四十八小时后，新鲜完整的RPMI 1640悬浮细胞在1 × 10 6细胞/ ml。为了诱使病毒生产，刺激细胞与20ng/ml tetradecanoyl佛波醋酸1小时（TPA）在一个标准的二氧化碳培养箱。用RPMI 1640洗涤细胞3次，以去除TPA。 </li><li>在原始卷的完全RPMI 1640（1.2）重悬细胞，并将其放置在二氧化碳培养箱96小时烧瓶。这种方法已被证明是产生高滴度的传染性病毒相提并论粒子6。 </li><li> 600 X克离心10分钟，4 ° C，从细胞中分离含有EB病毒培养上清。过滤上清液通过0.45微米的过滤器，分装，并存放于-70 ° C超过一年。 另一种方法是获得从B95 - 8细胞从ATCC（VR - 1492）中含有EB病毒上清，ATCC建议使用稀释。 </li></ol> 2。分离外周血单核细胞（PBMC）的<ol><li>从捐助绘制成肝素的注射器或肝素化血管10毫升血。在室温下稀释在50毫升的锥形管的血用20 ml PBS。 </li><li>底图稀释血液15毫升聚蔗糖Hypaque淋巴细胞分离液。离心225 × 克 ，在室温下30分钟的制动。 </li><li>删除捉鬼外套和转移到一个新的50 ml锥形管。量提高到50毫升，用PBS，在室温下在600 XG 10分钟旋转。 </li><li>宝R关闭上清。洗resuspending在50毫升PBS颗粒细胞，在室温和纺纱克600 × 10分钟。洗两次以类似的方式。 </li><li> 1毫升完全RPMI悬浮洗涤细胞。使用5μL细胞准备在台盼蓝的1 / 10稀释。使用一个血球计数活细胞。 </li><li>在一个25厘米2组织培养瓶使用完全RPMI获得了2 × 10 6 / ml的细胞浓度调节音量。 </li></ol> 3。感染EB病毒<ol><li>他克莫司（股份公司科学）的细胞悬液从2.6到终浓度为20纳米。置于容量瓶中， 二氧化碳培养箱在37 ° C为一小时。 </li><li>迅速解冻EB病毒等分。从孵化器中删除的烧瓶中，EB病毒添加到1 / 10稀释的细胞。通常情况下，这种稀释提供MOI 50-100。旋流烧瓶混合和直立放置在二氧化碳培养箱在37 ° C。 </li></ol>注意日在第4步进行预测成功的结果，是一个可选步骤。如果要执行第4步，你会需要孵育2 × 10 6细胞/毫升作为对照未感染者PBMC中的一个额外的烧瓶。 4。确定细胞增殖人口（可选步骤） <ol><li>准备流式细胞仪缓冲区：PBS + 5％FBS。保存在4 ° C。 </li><li>以下抗体各50μL的体积混合染色步骤4.6中的细胞。抗体稀释液应在流式细胞仪含1mg/ml的鼠IgG（Sigma公司）抑制非特异性结合的抗体的缓冲。 <ol class="lroman"><li>单染与PE标记的抗CD23抗体（1 / 50的稀释） </li><li>单染色FITC标记的抗CD58抗体（1 / 50的稀释） </li><li>单染与PE - Cy5的标记的抗CD19抗体（1 / 50的稀释） </li><li>三重染色CD23，CD58和CD19（每1 / 50稀释） </li><li>单PE标记的同型对照染色抗体相匹配（在相同浓度的抗CD23抗体CD23 - PE） </li><li>单染色FITC标记的同型对照抗体匹配（在相同浓度的抗CD58抗体CD58 - FITC） </li><li>单染色PE - Cy5的标记的同型对照抗体匹配，以CD19 - PE - Cy5的（在相同浓度的抗CD19抗体） </li><li>三，与所有三个亚型对照抗体染色。 </li></ol>在黑暗中暂时储存在4 ° C的混合物。 </li><li>在第三天后接触到EB病毒，轻轻漩涡容量瓶中，以获得更均匀的细胞悬液。卸下成2毫升Eppendorf管从烧瓶2毫升。自旋3分钟，在室温3000 RPM离心管。 </li><li>流式细胞仪缓冲吸上清液和悬浮细胞，在5 × 10 6 1 × 10 7细胞/毫升。 </li><li>删除8成单独的Eppendorf管50μL等分或96孔V型底板。旋管或盘在1500 × 克3分钟。 </li><li>丢弃上清和涡管/板。每管中的悬浮细胞/孔50μL流式细胞仪4.2编写包含相应的抗体稀释缓冲。 </li><li>就在30分钟的暗冰孵育管/板。在3分钟3000转离心细胞，取出上清液，加入200μL流式细胞仪缓冲。细胞再次离心，去除上清液完成洗。重复两个洗。 </li><li>悬浮细胞在200μL，流式细胞仪缓冲和采集数据，使用流式细胞仪，获得20000事件/管。 </li><li>使用WinMDI（流式细胞仪数据分析在PC免费）分析数据。门上使用正向和侧向散射配置的活细胞。然后，门活细胞表达CD19 +后与抗CD19抗体相匹配的同型对照抗体染色的细胞比较（B细胞标记）。剧情CD19 +生活乙Y轴X轴和CD58的荧光强度的细胞CD23的荧光强度。确定CD23 +和CD58 +细胞相匹配的同型对照抗体染色的细胞EB病毒暴露。 CD23 您好 CD58 +细胞（图2C）在EB病毒暴露的文化存在预测EB病毒感染的增长转化细胞的成功产物。相比之下，CD23 您好 CD58 +细胞不出现当细胞暴露于EB病毒（图2A）或接触到的EB病毒（图2B）非名垂千古的应变。 CD23 您好 CD58 +细胞已被实验证明经过扩散，并随后建立拼箱（14）。 </li></ol> 5。扩建和冷冻保存的拼箱<ol><li>可视光镜细胞：由EB病毒感染后一周，光镜下可见细胞簇。图3显示了一个早期微观集群的烧瓶中（图3B）的例子。 </li><li>随着时间的推移，微观集群成为较大等，团块可见宏观在烧瓶中。图3C建立拼箱细胞光镜显示更大的集群。 </li><li>喂养细胞：双12日在培养瓶中的培养液量。随后，扩大其体积用增加2-3倍，使用完全RPMI文化。 </li><li>喂养细胞周期的确定应基于细胞的生长速度。当培养基变成黄色，这是一般的时间，以取代上述媒体。通常情况下，发生这种情况，每周一次。然而，某些细胞株可能需要美联储或多或少频繁。在未来数周内展开文化75-100毫升。 </li><li>超低温冷冻保存：当冷冻细胞，离心600 × 克细胞在室温下10分钟。冷冰寒含90％FBS的培养基和10％二甲基亚砜取出上清，重悬细胞沉淀在1 × 10 7细胞/毫升。将管在液氮中。 </li></ol> 6。代表性的成果： 成功的结果是预测CD23 您好 CD58 +细胞的存在。大约2-3％的活细胞接触到EB病毒后3-4天，应该有此配置文件（图2C）。其他子群CD23 +，CD23 - ，CD58 +，和CD58 -细胞观察到EB病毒暴露后显示最小的扩散14。未感染的细胞（图2A）和暴露从HH514 - 16细胞（图2B），EB病毒窝藏非名垂千古的应变，EB病毒的细胞没有表现出CD23您好CD58 +细胞。 您也可以可视化细胞光镜监视成功的结果：正如前面提到的一个星期内，EB病毒感染后，烧瓶中的小群细胞是通过光镜（图3B）可见。随着时间的推移和细胞发展到拼箱，微观集群变大（图3C），团块肉眼可见烧瓶。 图1。淋巴细胞系的产生和冷冻保存的工作流程。外周血是通过聚蔗糖梯度离心。外周血单核细胞在一个既定的梯度巴菲大衣目前暴露FK506的EB病毒除了。在37 ° C在5％CO 2，建立和随后扩大为冻存拼箱存在EB病毒的细胞生长。 <img alt="图2" src="/files/ftp_upload/3321/3321fig2.jpg" /> 图2：一个预计接受暴露后，以EB病毒扩散的B细胞亚群的鉴定。联合国感染外周血单个核细胞（A）或外周血单核细胞暴露于EB病毒HH514 - 16细胞（B）或从B95 - 8细胞中存在的FK506（三）派生的第3天收获。细胞对CD19，CD23和CD58的荧光标记抗体染色。经过对活细胞的浇注S，未感染的（A）和EB病毒暴露（B和C），CD19 + B细胞CD23和CD58的表达研究。表达CD58和高水平的CD23（CD23 您好 CD58 +），B细胞的一个亚群指出，只有在细胞暴露改造主管EB病毒（B95 - 8细胞的），是描述。 <img alt="图3" src="/files/ftp_upload/3321/3321fig3.jpg" /> 图3。EB病毒感染细胞的细胞团的可视化。 PBMC中均采用FK506和与EB病毒感染。联合国感染（A）和EB病毒暴露（二）细胞研究的一个星期后感染相衬显微镜（10X放大倍率）。五老周淋巴母细胞线是在C

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没有利益冲突的声明。

本文中所描述的方法生成拼箱从捐助者外周血快速永生和冷冻保存时间。通过使用FK506的，我们是能够促进外周血单核细胞的EB病毒感染的B细胞的增殖T细胞的免疫抑制剂，和传染性病毒的高滴度。这些干预使描述更有效的方法，导致在随后的实验细胞快速扩张。 传统上，增长方式已经由约一个星期暴露后EB病毒6，15光学显微镜的细胞群的可视化监控。然而，细胞集群EB病毒介导的生长转型不是一个具体的指标。之前我们已经证实通过流式细胞仪检测14细胞增殖人口的一致鉴定，提供准确和具体的方法来确定成功的结果早3天船尾ER曝光B细胞的EB病毒。

<table border="1"><tbody><tr><td></td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>两性霉素B </td><td>费舍尔Bioreagents </td><td>血压928-250 </td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> ð 2650 </td></tr><tr><td>胎牛血清，热灭活</td><td>西格玛</td><td> F 4135 </td></tr><tr><td>聚蔗糖Hypaque淋巴细胞分离介质</td><td> Mediatech </td><td> 25-072 </td></tr><tr><td>他克莫司</td><td>公司科学</td><td> F 1030 </td></tr><tr><td>从小鼠血清抗体</td><td>西格玛</td><td>我8765 </td></tr><tr><td>鼠抗人CD23结合到PE </td><td> BD Pharmingen公司</td><td> 555711 </td></tr><tr><td>鼠标同型IgG1的结合到PE </td><td> BD Pharmingen公司</td><td> 554680 </td></tr><tr><td>鼠抗人CD58结合，以FITC标记</td><td>皮尔斯热SCientific </td><td> MA1 - 82159 </td></tr><tr><td>鼠标同型IgG1的结合，以FITC标记</td><td> BD Pharmingen公司</td><td> 550616 </td></tr><tr><td>鼠抗人CD19 PE - Cy5的标记</td><td> BD Pharmingen公司</td><td> 555414 </td></tr><tr><td>鼠标同型IgG1的PE - Cy5的共轭</td><td> Dako公司</td><td> X 0955 </td></tr><tr><td>青霉素/链霉素</td><td> Gibco公司</td><td> 15140-122 </td></tr><tr><td> RPMI 1640培养介质</td><td>西格玛</td><td> ř 8758 </td></tr><tr><td> Tetradecanoyl佛波酯（TPA） </td><td> Calbiochem </td><td> 407952 </td></tr><tr><td>流式细胞仪缓冲区（1X磷酸盐缓冲液+ 5％胎牛血清） </td><td></td><td></td></tr></tbody></table>

establishment of epstein-barr virus growth-transformed lymphoblastoid cell lines

B细胞与EB病毒（EBV）的感染导致扩散和随后的永生，致使建立的淋巴母细胞系在体外 （LCL） 。自拼箱潜伏EB病毒感染，他们提供了一个模型系统，以探讨EB病毒潜伏期，病毒驱动的B细胞增殖和肿瘤1。拼箱已用于目前在多种免疫检测2，3抗原。此外，拼箱可用于生成人类单克隆抗体4，5时，访问的主要生物材料是有限的6，7，提供了一个潜在的无限来源。 已被描述的各种方法，生成拼箱。较早的方法包括使用，如植物血凝素， 脂多糖 8有丝分裂原，美洲商陆有丝分裂原 （九）增加的EB病毒介导的永生化的效率。最近，其他人使用immunosuppr如环孢素essive代理商抑制T细胞介导的杀感染的B细胞7，10-12。 相当长的时间从EB病毒感染的细胞株的建立驱动器更快和更可靠的方法EBV驱动的B细胞生长转化的要求。使用高滴度的EB病毒和免疫抑制剂的组合，我们能够始终感染，改造，并从外周血B细胞产生拼箱。此方法使用少量的外周血， 在体外细胞集群感染的单核细胞可以证明。与EB病毒的存在，CD23，T细胞免疫抑制剂FK506的存在。传统上，B细胞增殖的生长约一周后与EB病毒感染的细胞的微观集群可视化监测。几个星期后肉眼可以看到的拼箱团块。我们描述了一个实验，以确定如果EB病毒介导克初rowth改造是成功的，即使在微观的细胞群可以证明。 您好 CD58 +细胞中观察到早3天，感染后的CD23的存在表明一个成功的结果。

Watch this Scientific Journal Video about Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines at JoVE.com

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

我们描述了一种用于发电使用EB病毒转化的B细胞株的方法。我们也说明了一种新的分析，可以识别注定要进行改造，早在感染后3天的B细胞。

Epstein - Barr病毒生长转化的淋巴细胞系的建立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

establishment-epstein-barr-virus-growth-transformed-lymphoblastoid

Research

JoVE Journal

Immunology and Infection

39.8K 次观看.  State University of New York at Stony Brook. 我们描述了一种用于发电使用EB病毒转化的B细胞株的方法。我们也说明了一种新的分析，可以识别注定要进行改造，早在感染后3天的B细胞。

视频: Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

胸腺淋巴瘤的T细胞系的推导 ATM - / -</sup P53 - / -</sup鼠标

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

科研

免疫与感染

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

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

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 inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma 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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...