这项工作得到了中国科学院重点项目 (KFZD-SW-205)、中国科学院战略生物资源技术支持系统 (CZBZX-1 和 ZSSB-004) 的支持, 并获得中国国家科学基金会资助 (81401640) 和上海自然科学基金 (14ZR1434800)。

这项研究获得中国科学院遗传学与发展生物学研究所伦理委员会的批准, 该议定书遵循人类福利的机构指南。
注意: 有关工作流的示意图, 请参见图 1 。
1. CAEBV 患者 PBMC 的分离
<ol><li>根据制造商的指示, 通过梯度 (例如, Ficoll 菌斑) 离心法从 CAEBV 患者的2毫升全血中纯化原 PBMC。或者, 用 RPMI 1640 培养基从液氮中解冻冷冻 PBMC。</li>
	<li>增加2µL 的台盼蓝 (0.4%) 到18µL 细胞悬浮, 等待3分钟染色细胞, 转移悬浮到细胞计数器板, 并测量细胞的浓度和活力与自动细胞计数器。</li>
	<li>准备细胞悬浮在密度 2-3×106细胞/毫升补充与完整的 RPMI 1640 培养基含有20% 人血清 (热灭活在56°c), 2 毫米 l-谷氨酰胺, 和抗生素 (100 µg/毫升链霉素, 100 U/毫升青霉素)。种子1毫升 PBMC 悬浮每井入24井细胞培养板材。</li>
	<li>每井加 150 U rhIL-2。</li>
	<li>将文化板块放置在 5% CO2孵化器中37摄氏度。</li>
	<li>2天, 观察显微镜下的细胞形态学 (200X 倍放大);细胞在明亮和圆的时候处于良好的状态。</li>
</ol>2. 细胞的扩张和可存活细胞的编号
<ol><li>观察细胞形态学在显微镜下 (200X 放大), 并监测细胞生长的条件, 通过编号的细胞改变培养基: 增加2µL 的台盼蓝 (0.4%) 到18µL 细胞悬浮, 并计算细胞的浓度 (见 1.2, 图 2)。</li>
	<li>在24井板中丢弃500µL 的培养基, 并添加500µL 新鲜完整的培养基。每井加 150U rhIL-2。</li>
	<li>按照步骤 2.2, 每周更改两次媒体。</li>
	<li>绘制单元格增长曲线 (图 3)。</li>
	<li>当存活细胞浓度超过 5×106/毫升时, 在每个井中, 将细胞分成 1-2×106细胞/ml 浓度的新井。这个过程需要2-4 周时间。</li>
</ol>3. 细胞分型流式细胞术
<ol><li>当单元格展开时, 收集 1×106单元格以确定单元格线的表型。240 x g的离心细胞在室温下为5分钟 (NK/T 细胞将沉淀在管的底部)。</li>
	<li>放弃上清, 通过添加7毫升 PBS 洗涤沉淀。在室温下, 离心机在 240 x g处为5分钟。重复一次。</li>
	<li>在每管中加入200µL 人血清, 混合均匀, 在室温下孵育10分钟。</li>
	<li>在室温下, 离心细胞在 240 x g 处为5分钟。丢弃上清, 然后在 1×106/mL 上用冷 PBSA (PBS + 0.2%BSA) 重新挂起单元格。将单元格分成5管, 每个管包含 2×105单元格。</li>
	<li>离心细胞在 240 x g 5 分钟在4摄氏度。丢弃上清, 再用含有10µL PE (或 PE-Cy7) 和10µL FITC 的 PBSA 缓冲或 PBSA 的细胞重新挂上标记的抗体, 如图 4所示, 将 T 或 NK 细胞的细胞受体染色。使用 PBSA 缓冲区暂停的单元格用作负控制。</li>
	<li>在黑暗中用20-30 分钟的冰块在冰上孵化细胞。</li>
	<li>用冷 PBSA 洗两次细胞, 用300µL 冷 PBSA 重新悬浮细胞。</li>
	<li>用流式细胞仪分析红细胞表型。<ol>
<li>在 "创建工作表" 条件下设置流式细胞仪。使用显示正散射 (FSC) 与侧面散射 (SSC) 的点图建立实验模板。</li>
		<li>负载同种控制管, 以优化 fsc 和 SSC 电压, 并优化 fsc 阈值, 以消除碎片, 而不干扰细胞群体的利益。删除所有参数, 除 FSC, SSC, FITC, PE 和 PE-Cy7。</li>
		<li>使用同种控件和每个2色分析组中的单个正则控件执行补偿。</li>
		<li>加载样本和创建 HLA-DR vs CD19, CD4 vs CD8, CD56 vs CD16 和 CD3 VS CD16 点地块显示不同的细胞数量。 注: PBMC 用于使用负/同种控制和单阳性控制进行补偿。采用流式细胞仪对2色免疫荧光进行了常规的表面标记表达分析。以下抗体包括: 抗 HLA-DR, anti-CD4, anti-CD16 共轭异硫氰酸荧光素 (FITC), anti-CD8, anti-CD56, CD3 与藻红蛋白 (PE) 共轭, anti-CD19 与 PE-Cy7 共轭。</li>
	</ol>
</li>
</ol>4. NK/T 细胞的扩展和冷冻保存
<ol><li>当细胞表型分析完成时, 改变培养基。小心地取出一半的上清, 避免接触盘子底部的细胞。添加相同数量的新鲜培养基中含有300毫升 rhIL-2 的细胞板。</li>
	<li>每3天改变一半的培养基 (2.2), 直到细胞簇在显微镜下清晰可见 (图 2)。通常, 此过程需要2-3 周时间。</li>
	<li>在混合同一血统的不同水井后, 将细胞从24孔板转移到 T25 培养瓶中。当培养基的体积扩大到10-15 毫升时, 将培养基的体积加倍, 使其变黄。添加 rhIL-2 的浓度为 150 U/毫升。</li>
	<li>改变培养基24小时后冷冻后2-3 周生长, 当细胞质量可以观察肉眼。用细胞计数器测量细胞浓度。</li>
	<li>离心细胞悬浮5分钟在 240 x g, 并重新悬浮细胞颗粒在密度为 5-10 x 106细胞/毫升与冷冻库存解决方案, 其中含有90% 胎牛血清和 10% dimethylsulfoxide (亚砜)。以每分钟1摄氏度的速率冻结至-80 摄氏度, 然后直接转入液氮。</li>
</ol>

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D.Z.、X.Z. 和 X.C. 是在等待专利申请方面的共同发明者, 涵盖了此方法用于建立 NK/T 细胞系的潜在用途以及本研究建立的细胞系。D.Z.、X.Z. 和 X.C. 宣布在这项工作中没有竞争的财政利益。其余的作者宣布, 他们没有竞争的金融利益。

在该协议中, 建立了 CAEBV 患者全血 NK/T 细胞系的新方法。与现有方法相比, 该方法的主要优点是其简单性和对少量血液的要求, 同时具有较高的成功率和良好的细胞生存条件。此外, 可以通过培养 PBMC 来建立 NK/T 细胞系, 而不确定 eb 病毒潜伏预先感染的细胞类型, 因为这种测定在培养前会消耗更多的血液和时间。另外, 细胞系可以通过细胞系建立后的流式细胞术进行分析, 从而纯化细胞的不同表型。?...

eb 病毒是无处不在的, 不仅感染了 B 细胞, 而且传染了 T 和自然杀手 (nk) 细胞, 这导致了一些 eb 病相关的 NK/T 淋巴疾病 (LPD) 和淋巴瘤/白血病, 如 eb 联合噬血细胞lymphohistiocytosis, hydroa vacciniforme 淋巴瘤, 淋巴结外 nk/T 细胞淋巴瘤, 鼻型和侵袭性 NK 细胞白血病 1, 2, 3.其中包括严重的慢性主动 eb 病毒 (SCAEBV) 疾病, 主要发生在东亚, 现在被认为是由 eb 病毒感染的 T 或 NK 细胞的克隆扩张引起的 LPD4,5,6,7, 但没有明显的免疫缺陷存在于传染性传染性 (IM) 类似的症状, 包括发烧, 肿大, 淋巴结肿大, 肝功能障碍持续或反复, 以及高 eb 病毒-DNA 负荷在外围血液8,9。CAEBV 患者预后不佳10,11, 其发病机制和 eb 病毒的作用尚不清楚。因此, 从 eb 病毒相关的 nk/t 淋巴疾病和淋巴瘤的细胞系是非常有用的细胞模型, 以澄清 eb 病毒诱发 NK 或 t 细胞增殖机制及其与高发病率的白血病或淋巴.
到目前为止, 已经用不同的技术建立了几个单元格线12,13,14,15,16。人类 nk 细胞系由 nk 细胞淋巴瘤/白血病建立, 通过与小鼠基质细胞系进行共培养, 并在 rhIL-2 的浓度为每毫升15。KAI3 是由患有严重蚊子过敏或 SCAEBV 的患者与自体永生细胞系 (拼箱)、乙肝病毒转化的 B 细胞、作为饲养细胞以及在 100 u/毫升16中添加 rhIL-2 的病人建立的另一种 NK 细胞系。SNK6 和 SNT8 通过添加大剂量的 rhIL-2 (700 u/毫升)12, 从鼻 NK/T 细胞淋巴瘤患者的肿瘤组织中获得。与类似的技术, SNK-1 细胞是从 CAEBV 患者, 培养从 PBMC 通过去除 T 细胞和添加 700 u/毫升 rhIL-213,17。SNT13 和 SNT15 是通过删除 CD4+ 和 CD8+ 单元格18建立的。到目前为止, 其他 t 细胞和 nk 细胞系从 eb 病毒-NK/T LPD 患者都开发了此方法19。
上述现有方法的弊端包括: 饲养细胞的使用、高剂量 IL-2 的要求、多达10毫升全血的利用率, 或对 NK/T 细胞的净化, 这在临床上是非常有挑战性的, 因为在开始培养之前, 必须验证 eb 病毒潜伏感染的细胞类型。由于 CAEBV 主要发生在亚洲儿童身上, 所以在所有地区都不容易获得10毫升血液。在本研究中, 我们开发了一种新的简单的方法, 成功率高, 建立 NK 和/或 T 细胞系, 通过培养 PBMC 从 CAEBV 患者使用低剂量的 rhIL2 和2毫升的全血容量, 没有喂食细胞。结果表明, 该方法效率高, 节省时间。

<table>
	<tbody>
		<tr>
			<td>Hu HLA-DR FITC</td>
			<td>BD</td>
			<td>560944</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu CD4 FITC</td>
			<td>BD</td>
			<td>555346</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu/NHP CD8 PE</td>
			<td>BD</td>
			<td>557086</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu CD3 PE</td>
			<td>BD</td>
			<td>555340</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu CD16 FITC 3G8</td>
			<td>BD</td>
			<td>555406</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>Hu/NHP CD56 PE MY31</td>
			<td>BD</td>
			<td>556647</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>hu/CD19 PE-Cy7</td>
			<td>BD</td>
			<td>560728</td>
			<td>antibody for FACS</td>
		</tr>
		<tr>
			<td>human IL-2</td>
			<td>Roche</td>
			<td>11147528001</td>
			<td></td>
		</tr>
		<tr>
			<td>human serum</td>
			<td>MRC</td>
			<td>CCC118-125</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>SANYO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Techcomp</td>
			<td>CT6T</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>OLYMPUS CKX53</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Countstar</td>
			<td>IC 1000</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>24 well cell culture cluster</td>
			<td>Corning Incorporated</td>
			<td>3524</td>
			<td>Polystyrene plates</td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Nest</td>
			<td>707001</td>
			<td>Polystyrene</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270</td>
			<td>Component of cell medium</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>life</td>
			<td>22400089</td>
			<td>For cell medium</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Amresco</td>
			<td>374</td>
			<td>Component of cell medium</td>
		</tr>
		<tr>
			<td>100 x streptomycin penicillin solution BioRoYeeBRY-2309</td>
			<td>BioRoYee</td>
			<td>BRY-2309</td>
			<td>Component of cell medium</td>
		</tr>
		<tr>
			<td>Ficoll paque plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td>For in vitro isolation of lymphocyte</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>For freezing cells</td>
		</tr>
		<tr>
			<td>flow cytometer</td>
			<td>BD</td>
			<td>BD FACSAria II</td>
			<td></td>
		</tr>
		<tr>
			<td>soft ware</td>
			<td>BD</td>
			<td>BD FACSDiva soft ware</td>
			<td>FACS analysis</td>
		</tr>
	</tbody>
</table>

an efficient and simple method to establish nk and t cell lines from patients with chronic active epstein-barr virus infection

从淋巴瘤或淋巴综合征患者中, 我们已经描述了一些方法来建立 NK/T 细胞系。这些方法使用饲养细胞, 纯化 NK 或 T 细胞多达10毫升的血液, 或高剂量的 IL-2。本研究提出了一种通过添加重组人 IL-2 (rhIL-2), 通过培养外周血单个核细胞 (PBMC) 来建立 NK 和 T 细胞系的新方法, 并使用2毫升的全血。细胞可以在两周内迅速增殖并维持3月以上。用该方法建立了 7 NK 或 T 细胞系, 成功率高。该方法简便、可靠, 适用于多例 CAEBV 或 NK/T 细胞淋巴瘤细胞株的建立。

在建立细胞系3天后, 多态细胞开始出现 (图 2)。7天后, 细胞生长迅速, 因为细胞的数量和生存能力都以较高的速率增加 (图 3)。当细胞浓度可超过 3-6 x 106时, 10-14 天后, 小簇细胞明显可见。在这一时期, 细胞应该被分成两到三口的培养板。大约一个月后, 一旦单元格号达到 3-5×107, 计数就足够高, 可以保存。
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Watch this Scientific Journal Video about An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection at JoVE.com

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

采用高效、少量外周血、低剂量 IL-2 的方法, 建立了 CAEBV 患者的 NK 和 T 细胞克隆的简便算法。

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 ...

an-efficient-simple-method-to-establish-nk-t-cell-lines-from-patients

Research

JoVE Journal

Immunology and Infection

9.1K 次观看.  Chinese Academy of Sciences. 采用高效、少量外周血、低剂量 IL-2 的方法, 建立了 CAEBV 患者的 NK 和 T 细胞克隆的简便算法。

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

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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

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

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

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 ...

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 ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.