Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining
淋巴细胞分离、Th17细胞分化、激活和染色
发布: 2016年12月05日第6卷第23期 DOI: 10.21769/BioProtoc.2047 浏览次数: 14607
评审: Ivan ZanoniAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2016
Abstract
In vitro Th17 (α, β T helper cell which produce IL-17A, IL-17F and IL-22) differentiation has been routinely used for functional T cells studies. Here we describe a method for Th17 cell differentiation.
Keywords: Th17 (Th17)
IL-17 (IL-17)
FACS (FACS)
Background
T cells are critical to mediate host defense against bacteria, viruses and fungi as well as commensal (Kumar et al., 2016). T cells can be further subdivided into T helper (Th1), Th2 and Th17 subsets based on their ability to generate specific cytokines. Naive T cells can be differentiated into specific T cell subsets in in vitro culture in response to specific cytokine stimulation. In vitro generated Th1, Th2 and Th17 cells have helped us to understand the molecular mechanism of their differentiation and their effector functions. Here, we have described a basic protocol for Th17 cell generation.
Materials and Reagents
- 96-well tissue culture plate (CELLTREAT Scientific Products, catalog number: 229196 )
- Falcon® 70 μm cell strainers (Corning, Falcon®, catalog number: 352350 )
- 50 ml conical tube (Denville Scientific, catalog number: C1056 )
- 1 ml syringe with cap (BD, catalog number: 305217 )
- 15 ml conical tube (Denville Scientific, catalog number: C1018-P )
- 96-well round (U) bottom plate (CELLTREAT Scientific Products, catalog number: 229190 )
- C57BL\6 mice (Taconic Biosciences, catalog number: B6-F )
- Coating antibodies: anti-mouse CD28 (Affymetrix, eBioscience, catalog number: 14-0281 ), anti-mouse CD3e (Affymetrix, eBioscience, catalog number: 14-0033 )
- ELISA coating buffer (Biolegend, catalog number: 421701 )
- EasySepTM buffer (STEMCELL Technologies, catalog number: 20144 ) or PBS containing 2% fetal bovine serum (FBS) with 1 mM EDTA
- EasySepTM Mouse Naïve CD4+ T Cell Isolation Kit (STEMCELL Technologies, catalog number: 19765 )
- Staining antibodies:
- anti-mouse CD3-eFlour 450 (Affymetrix, eBioscience, catalog number: 48-0032 )
- anti-mouse CD4-Alexa Flour 700 (Affymetrix, eBioscience, catalog number: 56-0041 )
- anti-mouse IL-22-APC (Affymetrix, eBioscience, catalog number: 17-7222 )
- anti-mouse IL-17A-PE (Affymetrix, eBioscience, catalog number: 12-7177 )
- anti-mouse CD62L-FITC (Affymetrix, eBioscience, catalog number: 11-0621 )
- Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
- Ionomycin (Sigma-Aldrich, catalog number: I0634 )
- Fixation/Permeabilization Solution Kit with BD GolgiStopTM (BD, catalog number: 554715 )
- Cytofix/Cytoperm Plus Kit (with BD GolgiStopTM) (BD, catalog number: 554715 )
- Iscove’s modified Dulbecco’s medium (IMDM) cell culture medium + glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 31980-030 )
- HyCloneTM fetal bovine serum (U.S.), characterized FBS (GE Healthcare, catalog number: SH30071.03 )
- HyCloneTM penicillin-streptomycin 100x solution (GE Healthcare, catalog number: SV30010 )
- Recombinant porcine TGF-β1 (R&D Systems, catalog number: 101-B1 )
- Recombinant mouse IL-6 (R&D Systems, catalog number: 406-ML )
- Recombinant mouse IL-23 (R&D Systems, catalog number: 1887-ML )
- Anti-mouse IFNγ (R&D Systems, catalog number: MAB485 )
- Anti-mouse IL-4 (R&D Systems, catalog number: MAB404 )
- Phosphate buffer saline (PBS) (Boston BioProduct, catalog number: BM220S )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3059 )
- Sodium azide (Sigma-Aldrich, catalog number: S2002 )
- Complete IMDM medium (see Recipes)
- 2x Th17 differentiation condition medium (see Recipes)
- FACS buffer (see Recipes)
Equipment
- Tissue culture incubator (NuAire, model: LabGard Class II type A2 )
- Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Legend XFR )
- BD LSRII Flow cytometer - BD
- Scientific Industries Vortex Genie2 (Stellar Scientific, catalog number: SI-236 )
- EasyEightTM EasySepTM magnet (STEMCELL Technologies, catalog number: 18103 )
Software
- FACS Diva or Flow Jo software
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kumar, P. and Kolls, J. K. (2016). Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining. Bio-protocol 6(23): e2047. DOI: 10.21769/BioProtoc.2047.
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分类
细胞生物学 > 细胞分离和培养 > 细胞分化
细胞生物学 > 细胞染色 > 全细胞
细胞生物学 > 基于细胞的分析方法 > 流式细胞术
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