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

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

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

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.

Abstract

腸内に付随して残りの侵襲的な病原体^1,2に向かって炎症反応をマウントする態勢を整えながら、共生植物や食物抗原に対する寛容の促進に関与している自然免疫と適応免疫細胞のユニークな集団を常駐します。抗原提示細胞、特に樹状細胞やマクロファージは、感知し、適切に細菌^3月14日に応答する能力を介して腸管免疫恒常性を維持する上で重要な役割を果たしている。腸の樹状細胞とマクロファージの効率的な分離は、これらの細胞の表現型と機能を特徴づける重要なステップです。樹状細胞とマクロファージを含む腸管免疫細胞を、単離の多くの効果的な方法は^{6,10,15-24を}説明してきたが、多くは否定的な細胞表面抗原の発現、細胞の生存、および/ または細胞収量に影響を与える可能性がある長い消化時間に依存しています。ここでは、詳細viabl大量の迅速分離するための方法論をE、腸の樹状細胞やマクロファージ。腸の樹状細胞とマクロファージの表現型の特性が直接マルチカラーフローサイトメトリー分析のために特定の蛍光標識モノクローナル抗体を用いて分離された腸の細胞を染色することにより行われる。さらに、高純度のDCおよびマクロファージの集団は細胞選別が続くとCD11b CD11cは磁気活性化細胞選別ビーズを利用した機能性研究のために隔離されています。

Protocol

1。腸上皮細胞の解離および解離試薬や機器の調製：室温へのCa 2 + / Mg 2 +のフリーのPBS（CMF PBS）暖かい。 5％FBS（CMF HBSS / FBS）と室温に2mMのEDTAと暖かいのCa 2 + / Mg 2 +のフリーのHBSS。 37暖かいオービタルシェーカー℃、注：ステップ1.1から1.7までは、細胞死の程度を最小限に抑?…

Discussion

図3。細胞収率と表面抗原の発現を最適化するための重要な要素。細胞収率と表面抗原の発現を直接組織消化の期間に影響される、コラゲナーゼの固有の特性、ミンチ組織の程度、炎症の有無、これは、組織の整合性と細胞性に影響を与える可能性があります。不十分な組織の消化は、分析のための細胞の不足につながるかもしれ?…

Declarações

The authors have nothing to disclose.

Acknowledgements

We thank Aaron Rae (Emory University Department of Pediatrics and Children’s Healthcare of Atlanta Flow Core) for cell sorting. This work was supported by NIH grant AA01787001, a Career Development Award from the Crohn’s and Colitis Foundation of America, and an Emory-Egleston Children’s Research Center seed grant to T.L.D.

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).