术研究采用由爱因斯坦癌症研究中心（NIH / NCI CA013330）和艾滋病研究中心（NIH / NIAID AI51519）的支持FACS核设施开展这项工作是由NIH / NIAID资助AI45889到SAP流程的支持。

动物实验是根据从体制动物护理和使用委员会（IACUC）批准的指导方针进行。所有需要无菌过程在生物安全柜进行。 1.小鼠黑色素瘤B16.Flt3L植入<ol><li>培养的Flt-3的表达B16黑色素瘤细胞中的T25组织培养瓶中以0.5×10 6细胞/ ml，在完全DMEM培养基中10％的CO 2的密度，在37℃线。生长，直到细胞达到〜90％汇合（约20小时）。 </li><li>由胰酶消化收获细胞。抽吸细胞培养基和洗涤贴壁细胞用PBS。添加5毫升0.05％胰蛋白酶并在37℃下孵育5分钟。添加冷20毫升完全DMEM培养基含有胎牛血清（FCS）（4℃）以猝灭蛋白酶消化。轻轻抬起关闭单元的割胶随后向下轻轻吹打起来。 </li><li>通过在300离心收集细胞xg离心在4℃下10分钟。算使用血球或自动化细胞存活率计数器细胞。悬浮于PBS中10 8个细胞/ ml的密度。 </li><li>通过皮下注射植入小鼠（C57BL / 6或另一组织相容性菌株）与肿瘤细胞如Machholz 等人 19在用100μl细胞悬浮液（10 7个细胞）的颈部的底部说明。 </li><li>让肿瘤生长，直到扪或可见牺牲动物器官收获前（2 - 1天 - 直径10毫米，一般为7）。 </li></ol>从荷瘤小鼠的脾单细胞悬液的2.准备工作<ol><li>麻醉小鼠异氟醚过量的（调整异氟醚的流量&gt; 5％，并继续呼吸停止暴露后至少2分钟）。根据安乐死的机构的指导方针颈椎脱臼牺牲FLT-3黑色素瘤轴承鼠标。 </li><li>迪sinfect使用无菌技术，用无菌剪刀20的辅助下，用70％的乙醇和收获脾皮肤。 </li><li>将脾脏进入无菌培养皿运输生物安全柜。在执行无菌条件下从这里的所有步骤。 </li><li>脾脏转移到一个新的培养皿中，并用RPMI培养基清洗。切脾为使用手术刀小（大约需要0.2 平方厘米）件。用10ml胶原酶/ DNA酶溶液温育30分钟，在37℃。 </li><li>倾脾组织的部分消化悬浮到70微米的细胞滤网。通过使用5毫升注射器柱塞过滤器的网眼压缩剩余组织碎片。 </li><li>用10ml新鲜培养基冲洗网状过滤器并丢弃滤波器。离心该悬浮液在300×g离心10分钟，在4℃以沉淀细胞。 </li><li>吸出上清液，重悬细胞沉淀在2ml RBC裂解缓冲液中。孵育RT˚F或10分钟。通过加入10ml的完全RPMI培养基猝灭RBC裂解缓冲液中。 </li><li>离心300 xg离心沉淀细胞在4℃5分钟。悬浮在适当体积以实现在含有20微克/毫升的Fc-块抗体（2.4G2）的细胞分选缓冲液（ 例如，MACS）的10 8个细胞/ ml的细胞密度。 </li></ol>从脾单细胞悬液浆的DC，CD8α 波什 DC和CD8α 负极性的DC 3.净化注意:从单细胞悬液脾DC亚群的分离是如在图1中所示的多步方法，用预冷却的缓冲液和冰水浴维持在4个温度下进行所有步骤- 8°C。在该协议的以下部分中的所有试剂体积计算为200微升脾细胞悬浮液以10 8细胞/ ml）的初始输入。这可以扩大或通过改变细胞悬浮液的体积（总是在1×10 8个细胞/ ml的密度）和其他试剂比例在初始步骤（3.1.1和3.2.1）向下根据你的需要。调整试剂卷因此以后所有步骤。例如，如果开始以1×10 8 / ml的100μl的脾细胞悬浮液，使用一半的所有步骤的所述试剂的卷。 <ol><li> 浆的DC隔离（PDC） <ol><li>加入300微升的浆细胞DC隔离套件提供给200微升脾细胞悬液，生物素标记的抗体混合物。 </li><li>调匀并在冰水浴孵育15分钟。轻按管每3分钟，以保持悬浮细胞，以最大限度地提高抗体结合。 </li><li>离心在300×g下上加12 ml的细胞分拣缓冲液和沉淀细胞5分钟，在4℃。吸去上清液，以除去未结合的生物素化的抗体。 </li><li>悬浮细胞沉淀在500微升细胞分选缓冲。添加300微升抗生物素抗体共轭珠。重复步骤3.1.2和3.1.3。 </li><li>弃上清，重悬细胞沉淀在2毫升细胞分选缓冲。执行使用预冷却缓冲器从这点上在RT的所有步骤。 </li><li>将新鲜的磁性细胞在磁力架排序LS列。洗并用5毫升细胞分拣​​缓冲液平衡柱。 </li><li>吸取细胞悬浮液上柱，轻轻将其应用于柱床的表面的中心。收集通过的流动。 </li><li>用10毫升细胞分拣​​缓冲液洗柱。通过收集该流并与流通过从步骤3.1.7结合。该pDC细胞富集通过（FT1）此列流动。 </li><li>在300 xg离心沉淀从FT1的细胞离心5分钟，在4℃下，悬浮在2ml细胞分拣缓冲液，并通过一个新鲜的LS柱通过细胞重复步骤3.1.6 - 3.1.8。这种使用两个连续列的产生分离细胞的纯度提高。 </li><li>沉淀细胞，在300×g离心5分钟，离心在4℃下，并重新悬浮用2ml完全RPMI。直到需要置于冰上。 </li></ol></li><li> CD8αPOS和CD8α 负极性的DC隔离 注意:在此过程中的第一步骤为B，PDC，T和NK细胞的耗竭。 <ol><li>与整个脾细胞悬浮液（1毫升在步骤2.8获得的10 8个细胞/ ml）开始，加入100微升的CD8α 方位直流隔离试剂盒中提供的生物素缀合的抗体混合物。 </li><li>拌匀并在冰水浴孵育15分钟。挖掘管轻轻每3分钟以生物素标记的抗体的结合最大化其靶细胞。 </li><li>离心300 XG上加12 ml的细胞分拣缓冲液和沉淀细胞5分钟，在4℃。超级吸游泳除去未结合的生物素化抗体。 </li><li>添加细胞分拣缓冲液150微升和100微升在CD8α 方位直流分离试剂盒提供抗生物素珠。拌匀并在冰水浴孵育15分钟。 </li><li>离心300 XG加入10 ml的细胞分拣缓冲液和沉淀细胞5分钟，在4℃。 </li><li>悬浮细胞在1ml细胞分拣缓冲，并根据在步骤3.1.6至3.1.8程序越过一个新鲜的LS柱。 </li><li>收集通过2（FT2）的未标记的流并丢弃柱滞留。此无标签部分富集了CD8αPOS和CD8α 负片的DC。 </li><li>在300 xg离心沉淀在FT2细胞离心在4℃下5分钟。 </li><li>重悬在1ml的细胞分选缓冲液中的细胞，并添加200微升的在CD8α 方位直流分离试剂盒提供抗CD8α偶联的磁珠。 </li><li>众议员吃步骤3.2.2通过3.2.6。通过柱的流动被富集CD8α 负片 DC和贫为CD8α 方位的DC。这是通过图3（FT3）标记的流动。 </li><li>为了洗脱CD8α 方位的DCs保留在柱中，从磁体取出柱，加入5 ml的细胞分拣缓冲液中，使用提供与LS柱柱塞吹扫柱基质。 </li><li>离心300 xg离心沉淀细胞在4℃5分钟。抽吸上清，重悬在1ml的细胞分选缓冲液中。为了提高纯度，通过一个新的LS列重复步骤3.1.6再次运行洗脱细胞3.1.8，除了放弃通过流量和收集留存细胞在3.2.11。 </li><li>从FT3悬浮液净化CD8α 负片的DC，沉淀通过离心FT3在300×g离心5分钟，在4℃下，并在300微升细胞分拣缓冲液中重悬。 </li><li>加入100微升抗CD11c MAG的NETIC珠。拌匀并在冰水浴孵育15分钟。 </li><li>离心300 XG加入10 ml的细胞分拣缓冲液和沉淀物的细胞5分钟，在4℃。 </li><li>重复步骤3.1.5至3.1.8。所述CD8α 负片的DC具有正的CD11c微珠选择并结合到柱上。流体通过可被丢弃。 </li><li>洗脱保留在柱中的细胞作为在步骤3.2.11收集纯化的CD8α 负片的DC说明。 </li></ol></li></ol> 4.脉动装甲运兵车与抗原和细胞转移<ol><li>使用计数台盼蓝21提纯区分活的和死细胞后活细胞。 </li><li>孵育选择的抗原的细胞。在100nM的浓度13使用糖脂抗原称为阿尔法半乳糖神经酰胺（αGalCer）类似物。通常，使用超低ATTAC板10 6活细胞/孔在完全RPMI培养基中hment U底96孔板，以尽量减少细胞附着。孵育在5％ 的 CO 2气氛中的细胞在37℃，1 - 4小时。 </li><li>收获细胞轻轻吹打数次。使用大口径的枪头，以尽量减少剪切力细胞损伤。用PBS洗井，集中这些洗涤以最大限度地提高电池的收获。 </li><li>沉淀细胞，在300×g离心5分钟，在4℃下重悬在PBS中期望的密度。通常情况下，注射1万个细胞中每个收件人动物200微升。保持冰的细胞给药前活力最大限度地发挥到​​宿主动物。 </li><li>注入细胞静脉内到小鼠无论是通过横向尾静脉或眶后静脉丛19。使用上的1ml注射器一个27克的针，并注入0.1ml的每只小鼠的最大体积。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

树突细胞接受是主要的专业抗原呈递参与T细胞应答的引发细胞。它们的主要功能是通过吸收和处理抗原呈现给T细胞来调查组织微环境。为了研究特定DC亚群的功能，这些需要使用保持其正常的表型和功能的方法在足够数量的分离。大多数协议依赖于特定DC亚群，从天真的小鼠隔离。因为这些细胞是罕见的，所述分离步骤是效率不高，并产生细胞的数量低。例如，一种脾将产生仅约0.5 -百万CD8 POS的DC，而用转移研究进行大多数实验至少使用这个号码每只动物的细胞。因此，现有的方法的一个主要局限性是不能在不使用供体小鼠和大量体积的非常大的数字来分离足够的细胞数量的试剂。我们的方法基本上克服了使用具有的Flt-3L分泌肿瘤植入到大大扩大的DC小鼠中，使用了一系列的免疫纯化步骤允许三种公认的DC子集的更大数量的纯化这些问题。这里所描述的协议，因此通过至少数倍提供在收率方面优于现有方法的重大改进。 几个注意事项此处描述的隔离协议存在。主要的限制是，用于分离这些细胞可能会无意中改变其表型的过程。 DC是内毒素污染和机械刺激，可能导致在它们的表型和功能的变化非常敏感。这种技术的另一个限制是对的Flt-3L分泌的依赖，以展开DC子集。这导致缺乏在不表达的Flt-3的受体组织驻留DC亚膨胀。 该这里描述的方案使用市售的试剂依赖于细胞富集。用于细胞纯化的分离的试剂盒附带包含试剂的推荐量要使用的协议。荷瘤小鼠的细胞组合物是从幼稚小鼠（ 图2C）的显着不同的，因此，我们已修改的量为，以达到最佳的细胞纯度在我们的技术中所使用的试剂。区议会是贴壁细胞，并可以连接到列矩阵的非特异性。用含EDTA缓冲液的柱床的平衡减少细胞粘附，并用连续的两列使用连续传代的进一步提高了细胞的纯度。 DC是高度贴壁细胞和体内通常紧密地与外组织基质相关联。为了提高产率，它与胶原酶和DNA酶足以恬消化脾组织是很重要的E要提取这些细胞的最大数量。同样重要的是要确保该脾组织片段在胶原酶/ DNA酶I溶液完全浸没在接受消化。直流隔离的另一关键特征是分离过程期间维持严格的无菌和无内毒素的条件下，因为这些细胞是高度内毒素敏感。从无内毒素的股票使用新鲜配制试剂和过滤器在使用前消毒所有试剂。所有的细胞分离缓冲和媒体应补充，经验证是免费的检测内毒素的FCS。这些细胞也对机械刺激和重复的机械刺激可以改变这些细胞的成熟状态高度敏感。虽然需要一些机械力通过胶原酶消化后细胞过滤迫使组织，这应该是尽量轻柔完成。另外，该细胞的有力的移液应避免多达possible和大口径枪头应该被用来最小化的剪切力。 过继转移模型是由DC的特定子集的抗原提呈细胞和T细胞引发的调查有用的。然而，已经证实在该转移的抗原可以从抗原致敏DC到内源性的DC发生的其他研究。因此，通过抗原刺激细胞策动适应性免疫应答可能反映被内源性的DCs呈递的"捕获"抗原，而不是该转移的APC的介绍。这种转移呈指数增加，如果隔离式DC制剂包含死亡或濒临死亡的细胞。它以尽可能快地进行DC隔离和传输协议是很重要的，使用具有预冷却的缓冲剂在无菌条件以增强细胞存活。我们选择了4小时的比较短的抗原脉冲期间为这个原因，在低结合细胞培养板孵育以最小化在细胞附着这一步。此外，扩展的培养条件可改变培养DC的成熟状态，并且可能影响的过继转移实验22的结果。 总之，树突状细胞生物学的研究非常活跃的领域，以及可靠的方法的发展，以产生的DC忠实地反映它们在体内的表型和功能分化提供了一个巨大的机会来研究这些细胞。这成为对于涉及可调节的DC功能，但不能被全身施用到动物模型的化学物质的研究尤其重要。治疗的兴趣体外用这些化学品的隔离式DC亚群是一个实际可行的途径，可以帮助阐明抗原呈递途径的机械细节。

树突状细胞（DC）近四十年前被发现的"大星状（希腊树枝 ）细胞"淋巴器官1中。许多研究表明，DC是，能有效地刺激幼稚T细胞2的唯一的抗原呈递细胞。这些细胞的主要功能是吸收和抗原的演示和高效的处理和这些装载到抗原呈递分子。在小鼠脾，DCS可以分离成浆和常规子集。该浆区议会由CD11c的低表达和高层次的B220和GR-1的表征。他们还积极为表面标志mPDCA1和分泌型我回应样受体9（TLR9）配体干扰素收费。传统的DC是很高的表面CD11c和MHC II类的表达。它们可以分成基于表型标记如CD4，CD8α，DEC205，CD的表面表达三种不同的亚群11b和树突细胞抑制性受体2（DCIR2由33D1抗体识别）的蛋白质3,4。所述CD8α 方位的DCs也称为CDC1，是阳性DEC205，但阴性骨髓标志物例如CD11b和33D1。该CD8α 负片的DC，也叫CDC2，都积极为33D1，CD11b和CD4但缺乏DEC205。双重否定的子集（ 即阴性CD4和CD8α）是比较少见的，而且是负DEC205和33D1。它是最特征子集，并且可以是CD8α 负片直流的较少分化形式。 在各个DC亚群的表型差异也延伸到他们的体内功能。所述CD8α 负片 DC是高度吞噬细胞和被认为呈现外源抗原主要通过MHC II类到的CD4 T细胞3。与此相反，CD8α 方位 DC是专门用在MHC I类可溶性蛋白抗原的介绍在一种机制称为交叉呈递。交叉呈递的结果取决于这些树突5的激活状态，并且可以导致细胞毒性T细胞（CTL）或调节性T细胞6 2,7-发展扩大。针对使用抗DEC205抗体介导的递送结果在很大程度上T细胞8的缺失抗原CD8α 波什的DC，而从感染的细胞凋亡细胞的抗原的演示诱导强CTL反应9。 除了识别肽抗原的，哺乳动物免疫系统已发展到识别脂质和糖脂抗原。这些抗原由CD1分子，其是存在于各种哺乳动物多个相关形式I类MHC样细胞表面蛋白呈现。在小鼠中，称为CD1d的高度保守的CD1分子的单一类型的负责糖脂抗原10的介绍。专业<html>识别CD1d分子/糖脂复合物的T细胞群被称为不变NKT细胞（iNKT细胞）。这些细胞表达了与TCRβ链具有有限多样性11配对的不变TCRα链组成的半不变的T细胞受体（TCR）。不像需要增殖和分化成为活化的效应T细胞的常规T细胞，iNKT细胞存在作为效应人口并开始糖脂给药12后迅速响应。生理相关脂质抗原呈递细胞的识别是一个活跃的研究领域，并且几种不同的细胞类型，例如B细胞，巨噬细胞和DC已经被建议来执行此功能。然而，有人证明DC的CD8α 方位子集是主要的细胞介导的摄取和脂质抗原呈现给小鼠iNKT细胞13和CD8 T细胞14的糖脂介导的交叉敏化。 ove_content"&gt;要由不同的抗原呈递细胞比较抗原呈递的效率，一个直接的方法是转移不同类型的抗原等量的脉冲进入幼稚主机。纯化的APCs这种类型的细胞转移实验通常为免疫学研究进行的。然而， 离体抗原处理的DC进行转移的研究是具有挑战性的，因为这些细胞存在于其中，它们构成总细胞15的小于2％的淋巴器官为罕见的人群。因此，有必要提高这些细胞的供体动物的发展增加的隔离协议的效率。 众所周知，在普通的淋巴和常见髓系祖细胞，其需要用于产生的pDC，CD8 方位和CD8 负片 DC亚群，快速FMS相关受体酪氨酸激酶3（FLT-3）的。 在体内的Flt-3配体（FLT-3L）给药，emigratFlt-3的表达来自骨髓祖细胞的离子增加，导致外周淋巴器官的增加的播种和它们的成熟的DC子代16的扩张。承诺B，T或NK细胞分化的途径中损失FLT-3的表达。因此，只有很少的改变是在这些细胞中时的Flt-3L给药观察到。在DC群相似膨胀荷瘤小鼠由B16黑素瘤细胞系分泌的鼠的Flt-3L，其中提供了用于提供的Flt-3L 17,18的持续全身水平的简单和经济的方法的注入而产生的肿瘤中观察到。使用这种方法，我们已开发了基于对B16黑色素瘤细胞的分泌的Flt-3L刺激小鼠脾所有正常DC亚群的膨胀，从而大大增加了这些细胞可被分离用于随后的实验的收率植入一个协议。我们一贯发现，在10 - 皮下IM14天在FLT-3分泌肿瘤的种植，小鼠树突状明显富集脾肿大发展构成40 - 总脾细胞的60％。从这些脾脏，不同的DC亚群可以使用标准的高纯度雇用特定子集，表型标记市售的细胞纯化试剂盒进行隔离。

<table><tbody><tr><td>0.05% Trypsin-EDTA&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>25300-054</td><td></td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CDS019936-250MG</td><td></td></tr><tr><td>Collagenase D&amp;nbsp;</td><td>Roche Diagnostics</td><td>11088858001</td><td></td></tr><tr><td>DNase I (dry powder)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>200 proof ethanol&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>9-6705-004-220</td><td>Used to prepare 70% ethanol</td></tr><tr><td>RBC lysis buffer</td><td>Sigma-Aldrich</td><td>R7757</td><td></td></tr><tr><td>RPMI-1640 medium with L-glutamine&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>11875-119</td><td></td></tr><tr><td>DMEM medium with L-glutamine&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>11995-073</td><td></td></tr><tr><td>200 mM L-glutamine&amp;nbsp;</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>MEM non-essential amino acids&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>11140-050</td><td></td></tr><tr><td>MEM essential amino acids&amp;nbsp;</td><td>Life Technologies</td><td>11130-051&amp;nbsp;</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Life Technologies, Invitrogen</td><td>21985-023</td><td></td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Life Technologies</td><td>11360-070</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies, Invitrogen</td><td>15630</td><td></td></tr><tr><td>Phosphate buffered saline (PBS Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; free pH 7.2)</td><td>Life Technologies, Invitrogen</td><td>20012-050</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s PBS (DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Life Technologies, Gibco</td><td>14040-182</td><td></td></tr><tr><td>0.5 M Ethylenediaminetetraacetate (EDTA) solution&amp;nbsp;</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Bovine serum albumin (BSA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Fetal calf serum</td><td>Atlanta Biologicals</td><td>S11050&amp;nbsp;</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Life Technologies, Invitrogen</td><td>15140-163</td><td></td></tr><tr><td>Trypan blue (dry powder)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6146-5G&amp;nbsp;</td><td>Prepare 0.08% with PBS</td></tr><tr><td>Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td><td>Miltenyi Biotech</td><td>130-092-786</td><td></td></tr><tr><td>Magnetic beads conjugated with anti-mouse CD11c&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-152-001</td><td></td></tr><tr><td>CD8&amp;alpha;&lt;sup&gt;Pos&lt;/sup&gt; mouse DC isolation kit&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-091-169</td><td></td></tr><tr><td>Fc-gamma receptor blocking antibody (Clone 2.4G2)</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>Anti-mouse CD11c-FITC&amp;nbsp;</td><td>BD Biosciences</td><td>553801</td><td></td></tr><tr><td>Anti-mouse CD8&amp;alpha;-PerCP&amp;nbsp;</td><td>BD Biosciences</td><td>553036</td><td></td></tr><tr><td>Anti-mouse B220-PE&amp;nbsp;</td><td>BD Biosciences</td><td>553090</td><td></td></tr><tr><td>1 ml syringes&amp;nbsp;</td><td>BD</td><td>26048</td><td></td></tr><tr><td>23 G1 needle&amp;nbsp;</td><td>BD</td><td>305145</td><td></td></tr><tr><td>100 mm Petri dishes&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>875712</td><td></td></tr><tr><td>Surgical instruments&amp;nbsp;</td><td>Kent Scientific</td><td>INSMOUSEKIT</td><td></td></tr><tr><td>Cell strainer (70 &amp;micro;m)</td><td>BD</td><td>352350</td><td></td></tr><tr><td>Large Petri plates&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Vacuum filtration system (500 ml (0.22 &amp;micro;m))</td><td>Corning</td><td>431097</td><td></td></tr><tr><td>LS columns&amp;nbsp;</td><td>Miltenyi</td><td>130-042-401</td><td></td></tr><tr><td>Magnetic stand MACS separator&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>Wide-bore 200 &amp;mu;l pipette tips&amp;nbsp;</td><td>PerkinElmer</td><td>111623</td><td></td></tr><tr><td>Corning ultra-low attachment 96-well plates&amp;nbsp;</td><td>Corning</td><td>CLS3474-24EA</td><td></td></tr><tr><td>6-8 week old female C57BL/6 mice&amp;nbsp;</td><td>Jackson Research Laboratories, Jax</td><td></td><td></td></tr><tr><td>Murine B16.Flt3L melanoma cell line</td><td></td><td></td><td>described by Mach &lt;em&gt;et al.&lt;/em&gt;, 2000</td></tr><tr><td>&lt;strong&gt;Serum free DMEM and RPMI media&lt;/strong&gt;&lt;br/&gt;500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine&lt;br/&gt;5 ml MEM nonessential amino acids (100x, 10&amp;nbsp;mM)&lt;br/&gt; 5 ml HEPES Buffer (1 M)&lt;br/&gt;5 ml L-glutamine (200&amp;nbsp;mM)&lt;br/&gt; 0.5 ml 2-mercaptoethanol (5.5 x 10&lt;sup&gt;-2&lt;/sup&gt; M)</td><td></td><td></td><td>Mix all the ingredients in a biosafety cabinet with either DMEM or RPMI media depending on your need. Filter sterilize the media by passing through a 0.22 &amp;micro;m vacuum filtration system.</td></tr><tr><td>&lt;strong&gt;Complete RPMI and DMEM media&lt;/strong&gt;</td><td></td><td></td><td>Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media.</td></tr><tr><td>&lt;strong&gt;MACS buffer&lt;/strong&gt;</td><td></td><td></td><td>Add 2 ml of 0.5 M EDTA and 10 ml of heat inactivated fetal calf serum to 500 ml of Phosphate-buffered saline (PBS, Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; Free, pH 7.2) and 2&amp;nbsp;&amp;micro;g/ml 2.4G2 (Fc-Block antibody).</td></tr><tr><td>&lt;strong&gt;FACS staining buffer&lt;/strong&gt;</td><td></td><td></td><td>Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer</td></tr><tr><td>&lt;strong&gt;10x Collagenase D and DNase 1 stock solution&lt;/strong&gt;&lt;br/&gt;
Dissolve 1 g of collagenase D and 0.2 ml of DNase 1 stock (1 mg/ml, 100x) in 20 ml of PBS containing Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; to obtain a solution of approximately 1,000-2,000 Units of collagenase activity per ml.</td><td></td><td></td><td>This 10x stock solution can be prepared ahead of time and stored at -20 &amp;deg;C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use.</td></tr></tbody></table>

an efficient and high yield method for isolation of mouse dendritic cell subsets

树突细胞（DC）是专业的抗原呈递细胞用于获取，处理和对抗原呈递分子以引发T细胞介导的​​免疫呈递的抗原主要责任。树突状细胞可被分离成多个表型上和功能上异质子集。脾树突状细胞的三个重要子集浆，CD8αPOS和CD8α 负片细胞。的浆DC是I型干扰素的天然生产者和用于抗病毒的T细胞免疫重要。该CD8α 负片 DC亚群是专门为MHCⅡ类抗原呈递并集中参与吸CD4 T细胞。该CD8α 波什的DC是外源性抗原和CD8 + T细胞引发的交叉呈现负主要责任。所述CD8α 方位的DC已被证明是最有效的在由CD1d的分子糖脂抗原的呈现给专用的T CEL被誉为不变的自然杀伤T（的iNKT）细胞L群体。的Flt-3配体的施用增加了从骨髓的树突状细胞前体的迁移的次数，最终导致在小鼠模型外周淋巴器官的树突状细胞的扩张。我们已经适应了这种模式来净化大量功能性树突状细胞在细胞转移实验使用比较体内不同的DC亚群的能力。

此程序的结果依赖于鼠的Flt-3L膨胀DC亚群由植入的黑色素瘤细胞中表达。该B16.Flt3L肿瘤是从C57BL / 6小鼠衍生的，而应该注入到动物与菌株背景中，以避免由于拒绝成为建立的肿瘤的故障。在某些情况下，可能需要使用转基因小鼠来推导在信号传导途径或感兴趣的受体与公知的缺陷的DC。重要的是要记住，肿瘤可能与在这些动物不同动力学发展是很重要的，所以根据外观和触诊在接种部位监测肿瘤生长是重要的。根据我们的经验，一旦肿瘤是显而易见的，在B16.Flt3L荷瘤小鼠一直显示在所有的DC亚群（ 图2A）的扩张相关的细胞结构脾明显增加。我们通常看到一个2到3倍，人口增加E在从相比幼稚动物B16.Flt3L黑素瘤携带小鼠获得的总脾细胞的数目。直流的部分数量的增加有助于B细胞的频率明显降低，而很少有在B细胞的绝对数量没有变化。有趣的是，当一个大的扩展（〜15中的绝对数量20倍的增加）被用于在这些小鼠中常规的DC观察到的，只有在浆细胞DC子集观察的适度增加（约4倍）。 选通策略，以确定不同的DC亚群示于图2A和基于B220，细胞CD11b，CD11c的和CD8α的具体子集的表型标记（ 图2B）中的表达。我们还研究DEC205，CD11b和mPDCA1表达对这些子集， 如图2B所示 。这表明仅在PDC子集的预期格局，mPDCA，而DEC<html> 205中高表达对CD8α 波什 DC和检测CD11b的最突出的CD8α 负片的DC。我们还分析在小区的频率的变化为每个在协议的每一步骤纯化子集这里描述（总结在图2C中 ，与图3中的流式细胞数据的详细示例）。在该方法中描述的脾DC亚群的分离是一个多步骤的过程（ 图1）。浆DC的纯化依赖于负选择所有不希望的人群像B，T，NK细胞和常规的DC枯竭后，以丰富这些细胞。所述CD8α 方位的DC，和CD8α 负片 DC的纯化通过这些细胞的阳性选择T，B和NK细胞的耗竭之后进行。使用此过程（通常为〜95％）在图3中还示出获得的各所述DC亚群的高纯度。 ve_content"FO:保持-together.within页="1"&gt;根据实验来进行，这些细胞可以用所需抗原是脉冲并转移到用于免疫学研究组织相容鼠主机此外，这些细胞也可以可用于体内的生理DC亚群的刺激或调节活性的研究。对于每2×10 7个总脾细胞的纯化的DC亚群的细胞产量是大约一百万的pDC，200万CD8α 方位的DC 400万CD8α 负片的DC。 <img alt="图1" src="/files/ftp_upload/53824/53824fig1.jpg" /> 图1. DC的隔离。说明该协议对发展中国家的不同子集隔离的各个步骤的示意图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53824/53824fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </a<html> &gt; <img alt="图2" src="/files/ftp_upload/53824/53824fig2.jpg" /> 图2.脾DC亚群的分析与B16.Flt3L肿瘤的小鼠。 A）的门控策略被示以识别对应于的pDC（R 1），CD8α 方位的DC（R 5）和CD8α 负片的DC（R 6）; B）mPDCA1，33D1，DEC205和CD11b的不同亚群表达的细胞群。包含在栅极R4中的细胞（淋巴细胞的子集;阴性B220，表面CD11c，CD11b的）被用作缺乏这些标记C）的直流的相对富集和淋巴细胞亚群从小鼠在一天的脾中分离的表达的对照群体。 B16.Flt3L黑素瘤细胞接种后14示于左侧情节作为黑条，而在幼稚小鼠这些细胞群的频率被示为白条。右侧图显示了相同的数据单元号。https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53824/53824fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53824/53824fig3.jpg" /> 图3.脾DC亚群分析富集协议期间。 A）从10％在从B16.Flt3L荷瘤小鼠的脾细胞的初始种群浆树突（B220 高和CD11c的低 ，R1）的富集至〜95％的纯度在柱流过。请注意，保留物从该柱洗脱的细胞从〜33％的脾脏耗尽这一人群。B）普通DC的富集（在高的CD11c，CD8α正面和负面的混合物）的B16.Flt3L黑色素瘤荷瘤植入后在〜78％，在第10天的小鼠通过2级分的流动。在列滞留2地块中，CD11c阳性的人群似乎是汉邦同盟耗尽，对应于负选择中去除DC的相当数量的耗尽T，B和NK细胞。通过2悬浮液使用阳性选择为抗CD8结合产率&gt; 95％纯度CD8α 方位 DC的流动进一步分级分离。然后通过从该流经受正选择的抗CD11c结合，产生&gt; 95％纯度CD8α 负片的DC人口。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53824/53824fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets at JoVE.com

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

an-efficient-high-yield-method-for-isolation-mouse-dendritic-cell

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

15.1K 次观看.  Albert Einstein College of Medicine. 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.

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

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

科研

癌症研究

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

生物学

Isolation of Mouse Lung Dendritic Cells

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 respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

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 ...

免疫与感染

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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 presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

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 ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

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

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

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 ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

神经科学

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Bone Marrow-Derived Dendritic Cells Generation: A Method to Generate Dendritic Cells from Mouse Bone Marrow

Source:Nguyen, H. D., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/60083">Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease.</a> J. Vis. Exp.. (2020).
This video describes a protocol for the generation of dendritic cells--key antigen-presenting cells that activate T cells--from the bone marrow of an adult mouse.

骨髓来源的树突状细胞生成:一种从小鼠骨髓生成树突状细胞的方法

Source:Nguyen, H. D., et al. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp.. ...