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

Duke Geem; Oscar Medina-Contreras; Wooki Kim; Clifton S. Huang; Timothy L. Denning

doi:10.3791/4040

JoVE Journal > Immunology and Infection

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Imunologia e Infecção

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Published: May 21, 2012

doi:

10.3791/4040

Duke Geem¹, Oscar Medina-Contreras¹, Wooki Kim², Clifton S. Huang¹, Timothy L. Denning^1,2

¹Department of Pediatrics,Emory University, ²Department of Pathology & Laboratory Medicine,Emory University

Summary

在这里，我们详细介绍了小鼠肠道树突状细胞（DCs）和巨噬细胞的快速分离方法。使用多色流式细胞仪分析细胞分选磁珠富集而被用来产生高纯度的功能研究人口肠道DC和巨噬细胞的表型特征。

Abstract

肠道内居住的是参与对共生的植物和食物抗原在促进容忍，而随之而来的剩余准备安装的炎症反应，病原体侵入朝^1,2先天免疫和适应性免疫细胞的独特群体。抗原提呈细胞，特别是区议会和巨噬细胞，发挥关键作用，在肠道免疫稳态维持通过自己的能力感和妥善应对微生物^3-14。肠道DC和巨噬细胞的高效分离是在描述这些细胞的表型和功能的关键一步。虽然隔离肠道免疫细胞，包括区议会和巨噬细胞，许多有效的方法已^{6,10,15-24，}许多依靠长期的消化时间，可能会产生负面影响细胞表面抗原的表达，细胞活力，和/或细胞产量。在这里，我们详细介绍快速分离的大量viabl的一种方法E，肠区议会和巨噬细胞。肠道DC和巨噬细胞的表型特征进行了直接染色孤立的肠细胞与特定的荧光标记单克隆抗体多色流式细胞仪分析。此外，高纯度的DC和巨噬细胞群体隔离功能的研究，利用CD11c和CD11b的磁激活细胞分选，细胞分选微珠。

Protocol

1。肠上皮细胞的解剖和离解制备的试剂和设备：温暖的Ca 2 + /镁2 +无PBS（CMF的PBS）的室温。温暖的Ca 2 + / Mg 2 +的免费的HBSS 5％胎牛血清（CMF方案的HBSS / FBS），和2mM EDTA室温。温暖的轨道摇床至37°C。注：步骤1.1至1.7，必须尽快进行，以尽量减少细胞死亡的程度，并实现最大的细胞?…

Discussion

图3。直接组织消化时间的影响因素，重要的细胞产量和表面抗原表达的优化。细胞产量和表面抗原表达，胶原酶的具体特点，组织切碎程度，以及存在或炎症的情况下，这可能会影响组织的完整性和细胞结构。长期组织消化，可能会导致细胞活力下降和表面抗原的表达，同时组织消化不足可能会导致在分析细胞缺乏。

…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢亚伦RAE（埃默里大学儿科和儿童医疗亚特兰大流核心），细胞分选。这项工作是由NIH资助AA01787001，从克隆氏病及溃疡性结肠炎美国基金会事业发展奖，和埃默里Egleston儿童研究中心种子批支持到顶级域名（TLD）

Materials

Name of the Reagent	Company	Catalogue number	Comments
1X PBS, Ca²⁺– and Mg²⁺-free
Hank’s balanced salt solution (HBSS) with phenol red	Fisher Scientific	SH3001603
Sodium bicarbonate	Sigma	S6014
1M HEPES in 0.85% NaCl	Lonza	17-737E
Fetal bovine serum (FBS)	Atlanta biologicals	S11150H	Heat-inactivated
0.5M EDTA (pH 8.0)	Cellgro	46-034-CI
Collagenase type VIII	Sigma	C2139
DNase I	Roche	14785000	Stock solution: 100mg/ml
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for 405 nm excitation	Invitrogen	L34957	Use at 1:1000
CD45-PerCP mAb (30F11)	BD	557235	Use at 1:100
CD103-PE mAb (M290)	BD	557495	Use at 1:100
FcγRIII/II mAb (2.4G2)	BD	553141	Use at 1:200
CD11c-APC mAb (N418)	eBioscience	17-0114-82	Use at 1:100
MHC-II (I-Ab)-Alexa Fluor 700 mAb	eBioscience	56-5321-82	Use at 1:100
CD11b-eFluor 450 mAb (M1/70)	eBioscience	48-0112-82	Use at 1:200
F4/80-PE-Cy7 mAb (BM8)	eBioscience	25-4801-82
CD11b microbeads	Miltenyi Biotec	130-049-601
CD11c microbeads	Miltenyi Biotec	130-052-001
50 mL conical tubes	BD Falcon	352098
Single mesh wire strainer	Chefmate
Small weigh boat	Fisher Scientific	08-732-116
100 μm cell strainer	BD Falcon	352360
40 μm cell strainer	BD Falcon	352340
5 mL polystyrene round-bottom tubes	BD Falcon	352235	Use at 1:100
MaxQ 4450 benchtop orbital shaker	Thermo Scientific
LS MACS column	Miltenyi Biotec	130-042-401
LSR II	BD
FACSAria II	BD

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Geem, D., Medina-Contreras, O., Kim, W., Huang, C. S., Denning, T. L. Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine. J. Vis. Exp. (63), e4040, doi:10.3791/4040 (2012).