体外诱导单核衍生的DC（树突状细胞）及其流式表型鉴定

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。而近年来的研究标明，单核细胞以依赖于细胞粘附分子CD62L和趋化因子受体CCR7的方式进入淋巴结，与直接从血液

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)。

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。

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【a. 人】

产品名称	厂商	货号
CD14 MicroBeads, human*	Miltenyi	130-050-201
Monocyte Isolation Kit II, human*	Miltenyi	130-091-153
MiniMACS Starting Kit §	Miltenyi	130-090-312
MidiMACS Starting Kit (LS) §	Miltenyi	130-042-301
autoMACS Running Buffer	Miltenyi	130-091-221-1.5
抗人CD14抗体	Miltenyi
*任选一种 §任选一种

【b.小鼠】

产品名称	厂商	货号
CD11b MicroBeads, human and mouse (130-049-601) *	Miltenyi	130-049-601
MiniMACS Starting Kit	Miltenyi	130-090-312
MidiMACS Starting Kit (LS)	Miltenyi	130-042-301
autoMACS Running Buffer	Miltenyi	130-091-221-1.5
抗小鼠CD11b抗体	Miltenyi

二、单核细胞的诱导培养及流式表型鉴定
1. 单核细胞诱导为未成熟DC或成熟DC

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【a. 人(6-10)】

产品名称	厂商	货号	规格
Human IL-4	PeproTech	200-04-5	5μg
	PeproTech	200-04-20	20μg
	PeproTech	200-04-50	50μg
	PeproTech	200-04-100	100μg
	PeproTech	200-04-250	250μg
	PeproTech	200-04-500	500μg
	PeproTech	200-04-1000	1000μg
Human GM-CSF	PeproTech	300-03-5	5μg
	PeproTech	300-03-20	20μg
	PeproTech	300-03-50	50μg
	PeproTech	300-03-100	100μg
	PeproTech	300-03-250	250μg
	PeproTech	300-03-500	500μg
	PeproTech	300-03-1000	1000μg
Human TNF-α*	PeproTech	300-01A-10	10μg
	PeproTech	300-01A-50	50μg
	PeproTech	300-01A-100	100μg
	PeproTech	300-01A-250	250μg
	PeproTech	300-01A-500	500μg
	PeproTech	300-01A -1000	1000μg
LPS*	sigma	L2880	10mg
抗人CD80抗体及同型对照	Miltenyi
抗人CD86抗体及同型对照	Miltenyi
抗人HLA-DR抗体及同型对照	Miltenyi
抗人CD83抗体及同型对照	Miltenyi
抗人CD40抗体及同型对照	Miltenyi
抗人CD1a抗体及同型对照	Miltenyi
TNF-α和LPS任选一种*

【b. 小鼠(11, 12)】

产品名称	厂商	货号	规格	目录价
Murine IL-4	PeproTech	214-14-5	5μg	960.00
	PeproTech	214-14-20	20μg	2340.00
	PeproTech	214-14-50	50μg	5460.00
	PeproTech	214-14-100	100μg	9100.00
	PeproTech	214-14-250	250μg	20475.00
	PeproTech	214-14-500	500μg	29575.00
	PeproTech	214-14-1000	1000μg	45500.00
Murine GM-CSF	PeproTech	315-03-5	5μg	960.00
	PeproTech	315-03-20	20μg	2340.00
	PeproTech	315-03-25	50μg	5460.00
	PeproTech	315-03-100	100μg	9100.00
	PeproTech	315-03-250	250μg	20475.00
	PeproTech	315-03-500	500μg	29575.00
	PeproTech	315-03-1000	1000μg	45500.00
LPS*	sigma	L2880	10mg	LPS*
抗小鼠CD11c抗体及同型对照	Miltenyi
抗小鼠CD40抗体及同型对照	Miltenyi
抗小鼠CD86抗体及同型对照	Miltenyi
抗小鼠CD11c抗体及同型对照	Miltenyi
抗小鼠I-Ab抗体及同型对照	Miltenyi

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