Name: isolation and adoptive transfer of high salt treated antigen-presenting dendritic cells
Uploaded: 2019-03-05T13:30:00
Description: Watch this Scientific Journal Video about Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells at JoVE.com

这项工作得到了美国心脏协会向 n. r. b.、17sdg33670829 至 l. x. 和国家卫生研究院向 a. k. 提供的 POST290900 赠款和 k01hl130497 的资助。

范德比尔特大学的机构动物护理和使用委员会已经批准了本文所述的程序。老鼠是根据《实验动物护理和使用指南》 (国家科学院出版社) 安置和照顾的。2010年修订)。
1. 从小鼠身上分离脾脏
<ol><li>制备 1640 rpmi:10% fbs、0.10 mm hepes、1 mm 丙酮酸钠、50μm β-硫醇和1% 青霉素。</li>
	<li>用二氧化碳吸入10–12周龄的 c57bl·6只雄性小鼠 。用70% 的乙醇喷洒小鼠的胸部和两侧。在?...

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在目前的协议中, 我们优化了从小鼠脾脏中分离 cd11c + dc的程序, 并将它们转移到天真的动物中, 以研究 dc 在盐性高血压中的作用。该方案可用于分离和采用转移其他免疫细胞亚群, 包括巨噬细胞、单核细胞和适应性免疫细胞, 包括 t 和 b 淋巴细胞。我们优化了脾脏消化过程, 以实现足够的细胞存活和稳定的直流表面表达标记。此外, 我们还优化了用钠升高体外刺激小鼠 dc 的方案, 以获得最佳...

过量的食盐是高血压的主要危险因素。1,2然而, 美国心脏协会建议每天最多摄入 2, 300 毫克钠 (na+);不到10% 的美国人遵守了这一建议。3 个,4适度降低 na+摄入量可降低血压, 并使美国每年新增的冠心病和中风病例减少20%。5过量食盐消费的一个主要问题是, 50% 的高血压人群表现出盐敏感性, 即 na+负荷后血压增加10毫米汞柱或 na + 后血压类似下降限制和利尿。6盐敏感性也发生在25% 的正常个体, 是死亡和心血管事件的独立预测指标。7.,8.对涉及肾脏的高血压的盐传感机制进行了很好的研究;然而, 最近的研究表明, 免疫细胞可以感觉到 na+。9,10
最近的证据表明, 肾外钠+处理的变化可导致间质中 na +的积累, 并促进炎症。11,12我们的实验室和其他实验室已经表明, 先天和适应性免疫系统的细胞都是导致高血压加重的原因之一。9,13,14,15各种高血压刺激, 包括血管紧张素 ii.、去甲肾上腺素和盐, 会引起巨噬细胞、单核细胞和 t 淋巴细胞浸润肾脏和血管, 促进 na+保留、血管收缩、血压高度和终末期器官损伤。9,16,17,18,19,20在先前的研究中, 我们发现 dc 积累了异丙基蛋白 (isolg)-蛋白质加合物, 以应对各种高血压刺激, 包括血管紧张素 ii. 和 doca-salt 高血压。14等长为脂质过氧化的高反应产物, 可迅速和共价地与赖氨酸结合在蛋白质上, 它们的积累与 dc 活化有关。14我们最近已经确定, 升高的 na+是对小鼠 dc 中等升蛋白的有效刺激. 9na +进入 dc 是通过氨基氯酰胺敏感转运体介导的.然后通过 na+/ca2 +交换器将 na+交换为钙 (ca2 +)。ca2 +激活蛋白激酶 c (pkc), 激活 nadph 氧化酶, 导致超氧化物 (o2·-) 和异丙基蛋白合管的形成。9含盐 dc 的收养转移是对血管紧张素 ii. 的反应的高血压的主要表现。9
以前, 从组织中识别 cd11c + dc仅限于免疫组织化学和 rt-pcr, 而 dc 的分离仅限于流式细胞仪的细胞分选。虽然流式细胞仪细胞分选是分离免疫细胞的有力方法, 但它成本高昂、耗时, 并导致活细胞产量低。因此, 我们对组织消化、体外刺激和采用 cd11c + dc转移的组织消化、体外刺激和采用转移来研究高血压进行了一步一步的优化。

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			<td height="21" style="height:21px;width:341px;">APC/Cy7 anti-mouse CD11c</td>
			<td style="width:160px;">Biolegend</td>
			<td align="right" style="width:140px;">117324</td>
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			<td height="21" style="height:21px;">autoMACS Running Buffer&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
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			<td height="21" style="height:21px;">CD11c MicroBeads Ultrapure&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-108-338</td>
			<td></td>
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			<td height="21" style="height:21px;">Collagenase D</td>
			<td>Roche</td>
			<td align="right">11088866001</td>
			<td></td>
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			<td height="21" style="height:21px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
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			<td height="21" style="height:21px;">DPBS without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
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			<td height="21" style="height:21px;">FcR Blocking Reagent</td>
			<td>Miltenyi Biotec</td>
			<td>&#160;130-092-575</td>
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			<td height="21" style="height:21px;width:341px;">FITC anti-mouse CD45</td>
			<td style="width:160px;">Biolegend</td>
			<td align="right" style="width:140px;">103108</td>
			<td></td>
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			<td height="21" style="height:21px;">GentleMACS C tube</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
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			<td height="21" style="height:21px;">GentleMACS dissociator device</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use protocol: Spleen 04.01</td>
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			<td height="21" style="height:21px;">LIVE/DEAD fixable violet dead cell stain kit</td>
			<td>Invitrogen</td>
			<td>L34964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuadroMACs Seperator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:140px;">11835-030</td>
			<td></td>
		</tr>
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isolation and adoptive transfer of high salt treated antigen-presenting dendritic cells

过量的膳食盐摄入会导致炎症, 并在高血压的发展中起着至关重要的作用。我们之前发现, 抗原呈现树突状细胞 (dc) 可以感觉到细胞外钠升高, 导致激活 nadph 氧化酶和形成异丙基蛋白 (异 lg)-蛋白导管。这些等脂蛋白加合物与自蛋白发生反应, 并促进自身免疫样状态和高血压。我们开发和优化了最先进的方法来研究高血压患者的直流功能。在这里, 我们提供了一个详细的方案, 隔离, 体外治疗升高钠, 并采用转移小鼠脾样 cd11c+细胞到接受者小鼠, 以研究他们在高血压中的作用。

图 1表示上述步骤的示意图。分离的小鼠脾脏通过磁性细胞分选进行分类,并在正常盐介质 (ns; 150 mmol ncl) 或高盐培养基 (hs; 190 mmol ncl) 中进行偏重基化细胞 + dc 的分类, 时间为 48小时. 然后通过反轨道通过 cd11c+ dc 进行收养转移注射到天真的受卵小鼠。十天后, 小鼠被植入渗透小细胞, 用于低剂量血管紧张素 ii (140 ng/kg/min) 输注14天。在14?...

Watch this Scientific Journal Video about Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells at JoVE.com

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

在这里, 我们提出了一个协议, 以分离树突状细胞从小鼠的脾脏通过磁性细胞分类和随后采用转移到天真的小鼠。选择高盐活化树突状细胞模型, 解释采用转移和流式细胞术的分步过程。

高盐抗原表达树突状细胞的分离和采用转移

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

该方法可以回答高血压和心血管疾病领域关键问题，包括CD11c阳性树突状细胞的研究，以及它们如何有效发挥高血压损伤的作用。该技术的主要优点是将CD11c阳性树突状细胞与固体器官分离，并研究它们在各种疾病状态中的生理作用。因此，这个过程可以应用于多种免疫细胞类型，包括B淋巴细胞，T淋巴细胞，单细胞和巨噬细胞。该技术的可视化对于了解树突细胞分离至关重要，特别是采用转移过程到接受小鼠。首先，用70%乙醇喷洒安乐死小鼠的胸部和侧面。在左侧，小心地打开皮肤和腹膜腔，露出脾脏。使用小钳子，小心地将肠子和肝脏移到小鼠的左侧。然后用小剪刀轻轻切除脾脏。将每个切除的脾脏放入一个标有 C 管的 C 管中，其中含有三毫升的 RPMI 1640 介质。首先将胶原酶D和DNase加入准备的RPMI 1640介质中，以准备脾脏消化液。将 C 管放在半自动均质器上，并确保将管子紧紧放在旋转臂上。按均质器上的启动按钮运行脾脏 04.01 协议 60 秒。在此之后，将管从均质器中分离出来，在 20 rpm 的连续温度下在 37 摄氏度下孵育样品 15 分钟。接下来，使用移液器将溶液通过 40 微米滤网过滤后转移到 50 毫升锥形管中。用 10 毫升 dPBS 清洗过滤器。在300倍g和4摄氏度下将细胞离心10分钟。然后完全吸气上一个液分，在 10 毫升 dPBS 中重新悬浮颗粒，然后清洗颗粒。在300倍g和4摄氏度下将悬浮液离心10分钟。将细胞颗粒重新悬浮在五毫升的 dPBS 中。使用血细胞计和 Trypan 蓝色排除数来计算细胞数。下一个离心机在300倍g和4摄氏度10分钟，以颗粒细胞。完全吸制上流液，并在400毫升磁激活细胞分选缓冲液中重新悬浮颗粒。然后每1亿个细胞加入100毫升CD11c微珠。在4摄氏度的冰箱中旋转细胞悬浮液并孵育10分钟。在此之后，加入10毫升的MC缓冲液，以300倍g和4摄氏度的温度下清洗细胞和离心机10分钟。完全吸升液，并在500毫升的MC缓冲液中重新悬浮细胞。将 LS 柱放在磁石分离器的磁场中，并在每根柱下放置一个 15 毫升的圆锥形管以收集流经。接下来，用三毫升的缓冲液冲洗柱子。将单元格悬浮到每一列上，并收集包含未标记单元格的流。用三毫升的MC缓冲液清洗柱子，并收集通过未标记的细胞。每次洗涤使用三毫升的 MC 缓冲液再清洗 LS 柱两次。在此之后，从磁性分离器中删除 LS 列。向每列添加五毫升的 MC 缓冲液，并立即将每根柱插入一个 15 毫升的透明圆锥管中，以冲出磁性标记的电池。使用血细胞计和 Trypan 蓝色排除确定文本协议中概述的 CD11c 正 DC 编号。然后在0.4%Trypan蓝色溶液中一对一稀释细胞样品。首先，在直流培养培养培养中准备细胞颗粒，如文本协议中所述。移液 900 微升直流悬浮成 24 孔平底猎鹰板。在每个井中加入100微升高盐DC培养培养剂，最终钠浓度为190毫摩尔。给盘子贴上标签，在37摄氏度的加湿二氧化碳培养箱中放置48小时。48 小时后，将板从培养箱中取出。将细胞培养培养培养器在一个井中上下移位，以重新挂起 CD11c 阳性DC。对镀井的每个井重复此项。将所有管以300倍g和4摄氏度离心10分钟。用 100 微升无菌 dPBS 吸升上一杯并重新悬浮直流颗粒。接下来，将直流溶液从其中一根管子中抽入一毫升注射器，并配有 27 量表半英寸针。将天真的男性10周大的C57-黑色-6小鼠放在2%异氟以下，实现稳定的手术平面。检查麻醉水平，缺乏踏板反射。一旦一个稳定的手术平面实现，慢慢地，小心地将针头引入反轨道空间，角度约为30度。缓慢而顺利地注入 CD11c 正直流悬架。注射完成后，小心地取出针对复古轨道空间，确保不损害眼睛。然后将小鼠从鼻锥中取出，并在麻醉恢复过程中监测小鼠约30分钟。一个典型的血压分析显示在这里后，采用转移DC和渗透迷你泵已被植入低剂量血管紧张素II输液。应该注意的是，这种低剂量的血管紧张素II是一种抑制剂剂量，不会增加正常小鼠的血压。接受正常盐处理CD11c阳性DC的小鼠在输液过程中维持正常血压，而接受高盐处理CD11c阳性DC的小鼠则表现出收缩压的增加。然后利用此处显示的浇注策略进行流式细胞学分析，以确定分离的 CD11c 阳性细胞的纯度。与总的飞溅细胞相比，CD11c阳性细胞的富集增加。不同的 CD11c 微珠浓度用于对制造商的协议进行故障排除。使用100微升微升微珠的飞毛细胞的磁性分离产生约65%的CD11c阳性细胞，而使用200微升产生55%，使用300微升产生50%的FcR阻滞剂和100微升的微升。磁分离后，FcR的封锁产生约65%CD11c阳性细胞。然后，一些飞毛细胞被孵育成100微升微升微珠，并通过LS柱进行磁分离。分离的细胞用CD11c的另外100微升孵育，并通过LS柱再次分离。这种双重隔离过程产生了92%CD11c阳性细胞。在尝试此过程时，通过流式细胞测量确认分离细胞的纯度非常重要。在树突状细胞进行磁隔离后，可以通过蛋白质组学和单细胞测序等分子技术培养它们以确定其活化状态。自其发展以来，现在可以研究树突状细胞在高血压中的作用和具体作用。研究人员正在探索高血压中特定的抗原。

Research

JoVE Journal

Immunology and Infection

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Isolation of Mouse Lung Dendritic Cells

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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

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

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

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

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

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...