这项研究得到香港研究资助局 (GRF 468513、CUHK3/CRF/12R) 及香港创新科技基金 (ITS/227/15、其 InP/164/16、ITS-InP/242/16)、中大 (2016.035) 及香港学者的直接资助。程序.
/设计和监督所有的实验, 并为手稿的编写做出贡献。康普茶进行了实验, 分析了数据并为稿件的编写做出了贡献。t。彼莱, 俱乐部, M.W., 和 G.-/收集动物标本, 并参与动物实验。右侧和 K-f.t 为手稿的编写做出了贡献。

获得和区分骨髓源性 nk 细胞的协议是基于以前发布的方法20,21,22。所有与老鼠有关的程序已获香港中文大学动物伦理实验委员会 (AEEC) 批准。
1. OP9 条件培养基的制备
<ol><li>培养小鼠基质细胞系 OP9 在α-记忆中含有 20% FBS, 100 U/毫升青霉素 G 和100µg/毫升链霉素, 在37° c 空气湿润大气/CO2 (95%: 5%)。<ol>
<li>用例计数单元格, 然后种子 2 x 106 OP9 细胞到每75厘米2培养瓶与15毫升培养基。文化为 48 h。</li>
		<li>当养殖达到80% 汇流时, 用10毫升 PBS 洗涤烧瓶, 并在每瓶中加入20毫升的普通α记忆培养基 (不含 FBS 和抗生素)。</li>
	</ol>
</li>
	<li>收集 OP9 条件培养基从培养烧瓶在24小时, 清理细胞碎片与0.25 µm 砜 (PES) 过滤器和保留在4° c (使用在2星期之内)。</li>
</ol>2. 分离小鼠骨髓细胞
<ol><li>安乐12周大的 C57BL/6J 鼠, pentobarbitone 过量 (100 毫克/千克, 腹腔注射)。确认死亡的缺乏呼吸, 然后收获股骨骨与手术刀切割的交界处之间的骨骼和删除剩余的肌肉与刀片的温柔划伤。</li>
	<li>将骨头放在一个50毫升的离心管中, 其中含有冷冻无菌的α记忆体, 然后在生物安全柜中执行以下步骤。</li>
	<li>丢弃α-记忆介质, 用70% 乙醇在三十年代冲洗骨头。</li>
	<li>用 ice-cold 消毒的 PBS 两次清洗骨头, 清理剩下的乙醇。</li>
	<li>将骨骼转移到含有5毫升 ice-cold PBS 的灰浆中, 用杵轻轻 fragmentize 骨头 (不要将其粉碎)。</li>
	<li>通过旋转杵来轻轻搅动骨骼, 将骨髓细胞释放到 PBS 中。将含有骨髓细胞的 PBS 收集到新的50毫升离心管中。</li>
	<li>重复步骤 2.6, 四次, 直到骨骼碎片变成纯白色的颜色。</li>
	<li>离心管在 465 x g 为5分钟在4° c, 然后放弃上清。</li>
	<li>重18毫升无菌蒸馏水, 并让它的立场三十年代, 以消除红血球。</li>
	<li>添加2毫升的 ice-cold 10X PBS 停止反应, 然后过滤的混合物通过一个70µm 细胞过滤器, 以消除裂解红血细胞。</li>
	<li>离心管在 465 x g 为5分钟在4° c。用20毫升的 ice-cold PBS 重颗粒。</li>
	<li>第二次重复步骤2.11 来清洗细胞。</li>
	<li>将细胞小球转移到100毫米的无菌培养皿中, 10 毫升的 OP9 条件培养基从步骤1.2 补充 20% FBS 和混合 0.5 ng/毫升小鼠 IL-7, 30 ng/毫升老鼠, 和 100 U/毫升鼠 flt3L。</li>
	<li>孵育盘在37° c 与 5% CO2为 2 h. 将未连接的单元格转移到新的区域性容器中, 其密度为 5 x 105可生存单元/mL (计数单元格例与台盼蓝排除) 与步骤2.13 中的相同介质公式, 并在37° c 下孵育 5% CO2。</li>
	<li>4天, 将培养条件转变为 OP9 条件培养基, 辅以 20% FBS 和2000小鼠 IL-2。</li>
	<li>每3天刷新培养基;成熟的 NK 细胞可在7天获得, 并符合4节的规定。</li>
</ol>3. siRNA 介导的分化 NK 细胞基因沉默
<ol><li>根据产品说明书将 siRNA 或无意义的控制 (NC) 与转染剂混合。</li>
	<li>从0天起, 在任何时间点执行步骤3.1。</li>
	<li>将 50 nM 的 siRNA 或 NC 混合物添加到步骤2.14 中的单元格。</li>
	<li>重复步骤3.1 在每个中等茶点 (例如, 天 0, 4 和 7), 直到实验结束点。</li>
</ol>4. 应用流式细胞仪分析 NK 细胞分化
<ol><li>在适当的时间点, 收集的悬浮分化细胞和洗涤与 ice-cold PBS。</li>
	<li>根据产品说明书将细胞固定缓冲液固定。</li>
	<li>用 ice-cold PBS 冲洗固定细胞, 然后在100µL 流式细胞术染色缓冲液中重 1 x 106细胞, 其中含有 Cy3-conjugated anti-mouse NKp46 和 PE 共轭 anti-mouse CD244 抗体, 稀释率为1:100。</li>
	<li>在室温下对样品进行2小时的染色。</li>
	<li>用 PBS 的300µL 重染色样品, 然后获取 Cytobank 平台20 (http://cytobank.org/) 的数据并进行分析。</li>
</ol>

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作者没有什么可透露的。

在目前的工作中, 我们描述了一种新的方法生产骨髓源性小鼠 NK 细胞体外。原始系统21、22中的单元格进纸器被 OP9 单元的条件介质成功替换, 这极大地提高了微分系统的稳定性。此外, 该系统还能为体外的成熟 nk 细胞产生高纯度的增殖和纯化, 并能促进人体疾病中 nk 细胞的机械研究和转化研究. 
所描述的方法已?...

癌症进展主要依赖于肿瘤微环境1,2, 包括主机派生的免疫,, NK 细胞。几项研究表明, 肿瘤的 NK 细胞呈负相关的癌症进展3,4。此外, 临床研究表明 NK 细胞过继疗法是一种可能的治疗癌症的策略5,6,7,8,9。NK 细胞癌免疫治疗是最近被认为是一种治疗选择的固体肿瘤, 但存在的挑战, 由于免疫抑制剂的分泌因子和下调激活配体在微环境中的实体肿瘤10,11。转化生长因子β (TGF β) 已被认为起抑制作用的致癌作用, 但矛盾的癌细胞也产生 TGF-β1支持肿瘤的发展12,13,14,15. TGF β信号通过下调干扰素反应性和 CD16-mediated 干扰素-γ-γ (干扰素) 产生抑制 NK 细胞的细胞活性体外16,17, 18。
虽然 TGF β信号在肿瘤微环境中的破坏可能是消除癌症的一种可能的方法, 完全阻断 TGF β信号将导致自身免疫性疾病, 由于其抗炎作用, 证明了发展不良副作用包括全身炎症、心血管缺陷和自身免疫在小鼠模型中的19。因此, 了解 TGF β介导的免疫抑制的工作机制将导致确定一个治疗癌症的辅助性治疗靶。
为了阐明 nk 细胞发育所需的分子事件, 威廉斯et al.建立了一个用于将小鼠骨髓造血干细胞分化为 nk 细胞的体外系统20。该系统在很大程度上促进了 nk 细胞发育的机制研究, 包括对 nk 细胞的新祖的鉴定21。然而, 骨髓祖细胞应在系统中培养, 以支持 OP9 基质干细胞为饲养层20,21, 而这种异构的细胞群在很大程度上限制了基因中断工具的进一步应用(例如, siRNA 介导的基因沉默) 专门用于区分 NK 细胞。
在这里, 我们描述一个无馈线系统, 已经开发的进一步修改的在体外系统的威廉姆斯et al20。在我们的系统中, 不需要 OP9 基质饲养细胞, 而使用 OP9 条件培养基, 而不影响 NK 细胞的分化在体外, 这最近导致我们发现, TGF β能够促进癌症进展通过抑制 E4bp4-dependent NK 细胞在肿瘤微环境中的发育22。该系统成功地提供了一种无背景的方法来阐明在特定条件下 NK 细胞发育的分子机制 (例如, 高 TGF β, siRNA 介导的基因沉默, etc。体外。

<table border="0" cellpadding="0" cellspacing="0" style="width:1113px;" width="1113">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">OP9 cell line</td>
			<td style="width:129px;">ATCC</td>
			<td style="width:344px;">ATCC&#174; CRL-2749&#8482;</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM &#945;, no nucleosides</td>
			<td>Gibco</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-7</td>
			<td>PEPROTECH</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine SCF</td>
			<td>PEPROTECH</td>
			<td>250-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine Flt3-Ligand</td>
			<td>PEPROTECH</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-2</td>
			<td>PEPROTECH</td>
			<td>212-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamin RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>1377815</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IC Fixation Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-8222-49</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow Cytometry Staining Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-conjugated anti-mouse CD244</td>
			<td>eBioscience</td>
			<td>12-2441-83</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cy3-conjugated anti-mouse NKp46</td>
			<td>Bioss</td>
			<td>bs-2417R-cy3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nonsense control (NC)</td>
			<td>Ribobio</td>
			<td>siN05815122147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">siRNA against mouse E4BP4 mRNA</td>
			<td>Ribobio</td>
			<td>N/A</td>
			<td>5&#8242;-GAUGAGGGUGUA 
			GUGGGCAAGUCUU-3&#8242;</td>
		</tr>
	</tbody>
</table>

a novel feeder-free system for mass production of murine natural killer cells in vitro

自然杀手 (NK) 细胞属于先天免疫系统, 是一线抗癌免疫防御;然而, 它们在肿瘤微环境中被压制, 其基本机制仍然是未知的。nk 细胞缺乏一致、可靠的来源, 限制了 nk 细胞免疫的研究进展。在这里, 我们报告一个体外系统, 可以提供高质量和数量的骨髓来源的小鼠 NK 细胞在无饲养条件下。更重要的是, 我们还证明, siRNA 介导的基因沉默成功地抑制了 E4bp4-dependent NK 细胞成熟, 利用该系统。因此, 这本新的体外NK 细胞分化系统是一种生物材料免疫研究的解决方案。

根据所述协议得到了代表性的结果。在无饲养诱导分化系统下培养的总骨髓悬浮细胞11天;7天, 与0天 (图 1A) 的总细胞数相比, 增殖率显著增加。在系统 (图 1B) 中, 6 天发现了成熟的 NK 细胞, 细胞核胞质比和富含颗粒的细胞质形态。
为了阐明 TGF-β1/Smad3 信号在 ...

Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

在这里, 我们提出了一个 mass-produce 基因沉默小鼠 NK 细胞的协议, 使用无馈线分化系统的机械研究体外和在体内。

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

a-novel-feeder-free-system-for-mass-production-murine-natural-killer

Research

JoVE Journal

Immunology and Infection

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

7.1K 次观看.  The Chinese University of Hong Kong. 在这里, 我们提出了一个 mass-produce 基因沉默小鼠 NK 细胞的协议, 使用无馈线分化系统的机械研究体外和在体内。

视频: A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

科研

免疫与感染

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

一种基于板的大鼠胎盘自然杀伤细胞细胞细胞溶解功能评价的细胞毒性测定

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

人类自然杀手细胞代谢分析

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...