Efficient Isolation of Influenza Specific CTLs
流感特异性的杀伤性T细胞的高效分离
发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1847 浏览次数: 9331
评审: Jia LiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
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Abstract
Human antigen-specific CD8+ T-cell clones are valuable tools for dissecting CD8+ T-cell responses against antigens derived from infectious agents, cancer and self antigens. Here we describe a protocol for isolating human antigen-specific CD8+ T cells. This protocol uses surface capture of IFNγ to identify antigen responsive cells that are then cloned by single-cell sorting. Here we use CD8+ T-cell responses to influenza matrix protein (MP) as an example, but this approach can be applied to any antigen specificity.
Keywords: Cloning (克隆)
CD8 (CD8)
CTL (CTL)
Human (人类)
T cell (T细胞)
Materials and Reagents
- 50 ml Falcon tubes
- Greiner CELLSTAR®, 96-well round-bottom tissue culture plates (Sigma-Aldrich, catalog number: M9311 )
- Corning® Costar® 24 and 48 well tissue culture plates (Sigma-Aldrich, catalog number: CLS3524 and CLS3548 )
- 10 ml tube
- Vials
- Peripheral blood
- EBV-transformed B-cell lines (HLA-A2)
- Ficoll (GE Healthcare, catalog number: 17-1440-03 )
- Phosphate buffered saline (DPBS) (Lonza, catalog number: 17-512F )
- Trypan blue solution (0.4%) (Sigma-Aldrich, catalog number: T8154 )
- RPMI 1640 (Sigma-Aldrich, catalog number: R8758 )
- Synthetic peptide(s) (Purar Chemicals)
- Influenza matrix peptide 58-66 (MP58-66; GILGFVFTL) (GL-Biochem)
- 5% pooled human serum
- Cytokines IL-2 (Peprotech, catalog number: AF-200-02 ), IL-15 (Peprotech, catalog number: AF-200-15 )
- Human IFNγELISA Kit (BioLegend, catalog number: 430106 )
- Miltenyi IFN-γ secretion assay kit-PE (Miltenyi Biotec, catalog number: 130-054-202 )
- CD8-FITC (SK1) mAb (clone SK1) (Thermo Fisher Scientific, eBiosciencesTM, catalog number: 11-0087-42 )
- Fungizone (Thermo Fisher Scientific, catalog number: 15290-018 )
- Propidium iodide (Sigma-Aldrich, catalog number: 81845 )
- Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
- Ionomycin (Sigma-Aldrich, catalog number: I0634 )
- Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
- HEPES (1 M) (Life technologies, GibcoTM, catalog number: 15630-080 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080”. - Non-essential amino acids (NEAA) (Life Technologies, GibcoTM, catalog number: 11140-050 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050”. - GlutaMAXTM (Life Technologies, GibcoTM, catalog number: 35050-061 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061”. - UltraPureTM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575-020 )
- Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P0781 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
- Stock concentration of cytokines and peptides (see Recipes)
- Cell culture media (see Recipes)
- Staining buffer (see Recipes)
- Stock concentration of propidium iodide (PI) (see Recipes)
- Stock concentration of PMA and ionomycin (see Recipes)
- Stock concentration of fungizone (see Recipes)
Equipment
- Megafuge 40R centrifuge (ThermoFisher, catalog number: 75004503 )
- Neubauer chamber
- Freezing container, Nalgene® Mr. Frosty (Sigma-Aldrich, C1562 )
- Cell culture CO2 incubator (Panasonic Healthcare Co., model: MCO-20AIC ) with 5% CO2
- Flow cytometer (BD Biosciences, model: BD FACSAria III )
- Liquid nitrogen dewar
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Trivedi, P. and Mannering, S. I. (2016). Efficient Isolation of Influenza Specific CTLs. Bio-protocol 6(12): e1847. DOI: 10.21769/BioProtoc.1847.
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分类
免疫学 > 免疫细胞功能 > 抗原特异反应
细胞生物学 > 细胞分离和培养 > 细胞分离
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