小鼠树突状细胞亚群的分离的高效和高产的方法
Summary
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.
Abstract
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。
Introduction
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“>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。
Protocol
Representative Results
Discussion
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 PO…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.05% Trypsin-EDTA | Life Technologies, Gibco | 25300-054 | |
| Isoflurane | Sigma-Aldrich | CDS019936-250MG | |
| Collagenase D | Roche Diagnostics | 11088858001 | |
| DNase I (dry powder) | QIAGEN | 79254 | |
| 200 proof ethanol | Thermo Fisher Scientific | 9-6705-004-220 | Used to prepare 70% ethanol |
| RBC lysis buffer | Sigma-Aldrich | R7757 | |
| RPMI-1640 medium with L-glutamine | Life Technologies, Gibco | 11875-119 | |
| DMEM medium with L-glutamine | Life Technologies, Gibco | 11995-073 | |
| 200 mM L-glutamine | Life Technologies | 25030081 | |
| MEM non-essential amino acids | Life Technologies, Gibco | 11140-050 | |
| MEM essential amino acids | Life Technologies | 11130-051 | |
| β-mercaptoethanol | Life Technologies, Invitrogen | 21985-023 | |
| Sodium pyruvate | Life Technologies | 11360-070 | |
| HEPES | Life Technologies, Invitrogen | 15630 | |
| Phosphate buffered saline (PBS Ca2+ and Mg2+ free pH 7.2) | Life Technologies, Invitrogen | 20012-050 | |
| Dulbecco’s PBS (DPBS with Ca2+ and Mg2+) | Life Technologies, Gibco | 14040-182 | |
| 0.5 M Ethylenediaminetetraacetate (EDTA) solution | Life Technologies | 15575-020 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A2153 | |
| Fetal calf serum | Atlanta Biologicals | S11050 | |
| Penicillin/streptomycin | Life Technologies, Invitrogen | 15140-163 | |
| Trypan blue (dry powder) | Sigma-Aldrich | T6146-5G | Prepare 0.08% with PBS |
| Plasmacytoid Dendritic Cell Isolation Kit II, mouse | Miltenyi Biotech | 130-092-786 | |
| Magnetic beads conjugated with anti-mouse CD11c | Miltenyi Biotech | 130-152-001 | |
| CD8αPos mouse DC isolation kit | Miltenyi Biotech | 130-091-169 | |
| Fc-gamma receptor blocking antibody (Clone 2.4G2) | BD Biosciences | 553141 | |
| Anti-mouse CD11c-FITC | BD Biosciences | 553801 | |
| Anti-mouse CD8α-PerCP | BD Biosciences | 553036 | |
| Anti-mouse B220-PE | BD Biosciences | 553090 | |
| 1 ml syringes | BD | 26048 | |
| 23 G1 needle | BD | 305145 | |
| 100 mm Petri dishes | Thermo Fisher Scientific | 875712 | |
| Surgical instruments | Kent Scientific | INSMOUSEKIT | |
| Cell strainer (70 µm | BD | 352350 | |
| Large Petri plates | Thermo Fisher Scientific | FB0875712 | |
| Vacuum filtration system (500 ml(0.22 um | Corning | 431097 | |
| LS columns | Miltenyi | 130-042-401 | |
| Magnetic stand MACS separator | Miltenyi Biotec | 130-042-302 | |
| Wide-bore 200 μl pipette tips | PerkinElmer | 111623 | |
| Corning ultra-low attachment 96-well plates | Corning | CLS3474-24EA | |
| 6-8 week old female C57BL/6 mice | Jackson Research Laboratories, Jax | ||
| Murine B16.Flt3L melanoma cell line | described by Mach et al. ,2000 | ||
| Serum free DMEM and RPMI media | |||
| 500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine 5 ml MEM nonessential amino acids (100x, 10 mM) 5 ml HEPES Buffer (1 M) 5 ml L-glutamine (200 mM) 0.5 ml 2-mercaptoethanol (5.5 x 10-2 M) |
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 µm vacuum filtration system. | ||
| Complete RPMI and DMEM media | Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media. | ||
| MACS buffer: | 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, Ca2+ and Mg2+ Free, pH 7.2) and 2 ug/ml 2.4G2 (Fc-Block antibody). | ||
| FACS staining buffer | Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer | ||
| 10x Collagenase D and DNase 1 stock solution 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++ and Mg++ to obtain a solution of approximately 1,000- |
2,000 Units of collagenase activity per ml This 10x stock solution can be prepared ahead of time and stored at -20 °C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use |
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