文章目录[隐藏]
简介
DC细胞是体内最有效的抗原递呈细胞(APC)，可起始幼稚T细胞，并维持特异性T细胞对肿瘤或病原体的应答(1)。DC以未成熟的状态存在于在大多数组织中，可高效地摄取抗原。在遇到外来抗原时，DC会经历复杂的成熟过程，并成为专职的APC，细胞表面的组织相容性复合物(MHC) I和II类以及共刺激分子CD80、CD86和CD40均会表达上调。与此同时，DC的抗原捕获机制下调(1)。此外，DC细胞表面的受体表达发生变化，改变其对各种趋化因子的应答，从而通过输入淋巴管从组织进入局部淋巴结的皮质旁区(2)。低频的抗原特异性幼稚T细胞或记忆性T细胞在此遇到这些负载抗原的DC细胞时，可识别MHC-多肽复合物，从而起始特异性免疫应答。
虽然DC分布广泛，但其比例非常低，并且难以分离，无法获得大量高纯度的DC细胞(3)。在体外将单核细胞诱导为DC细胞是目前研究DC分化和成熟非常有效的方法(4)。这些单核衍生的DC细胞(Mo-DC)可高效捕获可溶性抗原，对微生物和炎症刺激物产生应答。小鼠单核细胞在对脂多糖(LPS)或革兰氏阴性菌产生应答，迁移至外周淋巴结，迅速获得DC细胞的关键特性，如形态和将外源性抗原递呈给T细胞的能力(5)。
实验步骤
一、单核细胞纯化
应用免疫磁珠从人或小鼠的外周血或淋巴组织中分离高纯度的单核细胞。
人单核细胞纯化试剂列表
| 产品名称 | 厂商 | 货号 | 目录价￥ |
| CD14 MicroBeads, human* | Miltenyi | 130-050-201 | 9016.00 |
| Monocyte Isolation Kit II, human* | Miltenyi | 130-091-153 | 15258.00 |
| MiniMACS Starting Kit § | Miltenyi | 130-090-312 | 10485.00 |
| MidiMACS Starting Kit (LS) § | Miltenyi | 130-042-301 | 20867.00 |
| autoMACS Running Buffer | Miltenyi | 130-091-221-1.5 | 877.00 |
| 抗人CD14抗体 | eBioscience | / | / |
*任选一种 §任选一种
小鼠单核细胞纯化列表
| 产品名称 | 厂商 | 货号 | 目录价(￥) |
| CD11b MicroBeads, human andmouse (130-049-601) * | Miltenyi | 130-049-601 | 11804.00 |
| MiniMACS Starting Kit | Miltenyi | 130-090-312 | 10485.00 |
| MidiMACS Starting Kit (LS) | Miltenyi | 130-042-301 | 20867.00 |
| autoMACS Running Buffer | Miltenyi | 130-091-221-1.5 | 877.00 |
| 抗小鼠CD11b抗体 | eBioscience | / | / |
*为获得高纯度的单核细胞，可先用CD11c (Miltenyi，130-052-001)、Ly-6G (Miltenyi，130-092-332)和CD49b (DX-5) (Miltenyi，130-052-501)磁珠分别去除DC、粒细胞、NK细胞。
二、 单核细胞的诱导培养及流式表型鉴定
单核细胞诱导为未成熟DC或成熟DC
【a. 人单核细胞的诱导及流式表型鉴定】
| 产品名称 | 厂商 | 货号 | 规格 | 目录价 |
| Human IL-4 | PeproTech | 96-200-04-5 | 5μg | 960.00 |
| PeproTech | 96-200-04-20 | 20μg | 2340.00 | |
| PeproTech | 96-200-04-50 | 50μg | 5460.00 | |
| PeproTech | 96-200-04-100 | 100μg | 9100.00 | |
| PeproTech | 96-200-04-250 | 250μg | 20475.00 | |
| PeproTech | 96-200-04-500 | 500μg | 29575.00 | |
| PeproTech | 96-200-04-1000 | 1000μg | 45500.00 | |
| Human GM-CSF | PeproTech | 96-300-03-5 | 5μg | 960.00 |
| PeproTech | 96-300-03-20 | 20μg | 2340.00 | |
| PeproTech | 96-300-03-50 | 50μg | 5460.00 | |
| PeproTech | 96-300-03-100 | 100μg | 9100.00 | |
| PeproTech | 96-300-03-250 | 250μg | 20475.00 | |
| PeproTech | 96-300-03-500 | 500μg | 29575.00 | |
| PeproTech | 96-300-03-1000 | 1000μg | 45500.00 | |
| Human TNF-α* | PeproTech | 96-300-01A-10 | 10μg | 960.00 |
| PeproTech | 96-300-01A-50 | 50μg | 2340.00 | |
| PeproTech | 96-300-01A-100 | 100μg | 5250.00 | |
| PeproTech | 96-300-01A-250 | 250μg | 9450.00 | |
| PeproTech | 96-300-01A-500 | 500μg | 13650.00 | |
| PeproTech | 96-300-01A -1000 | 1000μg | 21000.00 | |
| LPS* | MultiSciences | CS0006 | 0.5mg | 90.00 |
| 抗人CD80抗体及同型对照 | eBioscience | / | / | / |
| 抗人CD86抗体及同型对照 | eBioscience | / | / | / |
| 抗人HLA-DR抗体及同型对照 | eBioscience | / | / | / |
| 抗人CD83抗体及同型对照 | eBioscience | / | / | / |
| 抗人CD40抗体及同型对照 | eBioscience | / | / | / |
| 抗人CD1a抗体及同型对照 | eBioscience | / | / |
*TNF-α和LPS任选一种
【b. 小鼠单核细胞的诱导及流式表型鉴定试剂列表】
| 产品名称 | 厂商 | 货号 | 规格 | 目录价 |
| Murine IL-4 | PeproTech | 96-214-14-5 | 5μg | 960.00 |
| PeproTech | 96-214-14-20 | 20μg | 2340.00 | |
| PeproTech | 96-214-14-50 | 50μg | 5460.00 | |
| PeproTech | 96-214-14-100 | 100μg | 9100.00 | |
| PeproTech | 96-214-14-250 | 250μg | 20475.00 | |
| PeproTech | 96-214-14-500 | 500μg | 29575.00 | |
| PeproTech | 96-214-14-1000 | 1000μg | 45500.00 | |
| Murine GM-CSF | PeproTech | 96-315-03-5 | 5μg | 960.00 |
| PeproTech | 96-315-03-20 | 20μg | 2340.00 | |
| PeproTech | 96-315-03-25 | 50μg | 5460.00 | |
| PeproTech | 96-315-03-100 | 100μg | 9100.00 | |
| PeproTech | 96-315-03-250 | 250μg | 20475.00 | |
| PeproTech | 96-315-03-500 | 500μg | 29575.00 | |
| PeproTech | 96-315-03-1000 | 1000μg | 45500.00 | |
| LPS | MultiSciences | CS0006 | 0.5mg | 90.00 |
| 抗小鼠CD11c抗体及同型对照 | eBioscience | / | / | / |
| 抗小鼠CD40抗体及同型对照 | eBioscience | / | / | / |
| 抗小鼠CD86抗体及同型对照 | eBioscience | / | / | / |
| 抗小鼠CD11c抗体及同型对照 | eBioscience | / | / | / |
| 抗小鼠I-Ab抗体及同型对照 | eBioscience | / | / | / |
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