T Follicular Helper Cell Coculture Assay
滤泡性辅助性T细胞（Tfh）与B细胞共培养系统
发布: 2014年01月05日第4卷第1期 DOI: 10.21769/BioProtoc.1021 浏览次数: 16912
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
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Abstract
T follicular helper (Tfh) cells constitute a distinct subset of CD4+ T cells specialized in providing help to B cells in germinal centers. Phenotypically, Tfh cells are characterized by their high expression of the chemokine receptor CXCR5 that allows their migration into B cell follicles as well as high expression of PD-1, BTLA, the co-stimulatory molecules ICOS and SLAM and the transcription factors BCL6 and cMaf. Tfh cells are the main producers of IL-21 as well as other cytokines like IL-4 and IL-10 critical for B cell survival and differentiation. Tfh cells drive somatic hypermutation and the generation of long-lived memory B cells and plasma cells having an essential role in the development of protective immunity. Developing a coculture system to measure the effects of Tfh-cell mediated B cell help is of great interest to further our understanding of Tfh-B cell interaction and to allow for the manipulation of culture conditions to investigate the potential effect different microenvironment signals or ligand/receptor interactions could have on Tfh cell function.
Materials and Reagents
- Lymph node mononuclear cells (LMNCs)
- Benzonse® Nuclease (EMD Millipore, catalog number: 70664 )
- LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Life Technologies, catalog number: L34957 )
- Staphylococcal enterotoxin B (Toxin Technology Inc, catalog number: BT202 )
- RPMI 1640 Medium 1x with L-Glutamine (Fischer Scientific, catalog number: 10-040-CV )
- 100x Penicillin-Streptomycin solution (5000 units) (Life Technologies, catalog number: 15070063 )
- Fetal Bovine Serum (FBS) (Access Cell Culture)
- RPMI 1640 Medium 1x with L-Glutamine and no Phenol red (Gibco®, Life Technologies, catalog number: 11835-030 )
- HEPES solution (1 M) (Sigma-Aldrich, catalog number: H0887 )
- Antibodies:
- Complete media (see Recipes)
- Sorting buffer (see Recipes)
Equipment
- Centrifuge (SorvallTM LegendTM XTR, Thermo Fisher Scientific)
- 15 ml Falcon tube
- BD FACSAria II cell sorter (BD Biosciences)
- 37 °C 5% CO2 incubator
- 96-well sterile V-bottom plates (Thermo Fisher Scientific, catalog number: 249935 )
- 5 ml polystyrene round-bottom tubes with cell-strainer cap (BD FalconTM, catalog number: 352235 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Cubas, R. A. and Haddad, E. K. (2014). T Follicular Helper Cell Coculture Assay. Bio-protocol 4(1): e1021. DOI: 10.21769/BioProtoc.1021.
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分类
免疫学 > 免疫细胞分离 > 淋巴细胞
细胞生物学 > 细胞分离和培养 > 细胞生长
免疫学 > 免疫细胞功能 > 淋巴细胞
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