Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction
建立可同时研究T淋巴细胞增殖和树突细胞极化的共培养体系
发布: 2016年03月05日第6卷第5期 DOI: 10.21769/BioProtoc.1750 浏览次数: 11784
评审: Ivan ZanoniRamalingam BethunaickanBenoit Chassaing
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
Abstract
Dendritic cells (DCs) play a critical role in mounting the T cell response against different infectious agents. Nature and intensity of the induced T cell responses are defined by activation status of DCs. It is generally accepted that IL-12, IL-4/IL-5 and IL-23 producing DCs induce TH1, TH2 and TH17 type of immune responses, respectively (Kumar et al., 2015). Besides cytokines, levels of co-stimulatory molecules on DCs also influence the response of T cells.
The activation status of DCs can be determined by examining DC culture supernatants for different cytokines and by analyzing expression of co-stimulatory molecules on these cells. However, these approaches provide indirect information about T cell activating potential of DCs. Analysis of T cell responses in a co-culture system is a more direct approach to examine T cell proliferating and polarizing efficacy of DCs.
A protocol to analyze the T cell proliferating and polarizing potential of DCs in an allogeneic mixed leukocyte reaction (allo-MLR) is described here.
Materials and Reagents
- RPMI-1640 medium (HiMedia Laboratories, catalog number: AT028 )
- Dulbecco’s Phosphate Buffered Saline (HiMedia Laboratories, catalog number: TS1006 )
- Heat-inactivated fetal bovine serum (Biological industries, catalog number: 04-121-1A )
- Antibiotic-antimycotic (penicillin-streptomycin) solution, 100x (HiMedia Laboratories, catalog number: A002A )
- Round bottom, 96-well cell culture plates (Corning, catalog number: 3799 )
- Dendritic cells (derived by culturing mouse bone marrow cells in the presence of recombinant GM-CSF) (PeproTech, catalog number: 315-03 )
- Untouched CD4+ and CD8+ T cells from allogeneic mouse strain (isolated from spleen of Balb/c mice using CD4 T cell enrichment kit and CD8 T cell enrichment kit (BD, catalog number: 558131 and 558471 , respectively)
- Fluorochrome-conjugated FITC anti-mouse CD3, PE anti-mouse CD4 and PE anti-mouse CD8 antibodies (BD Pharmingen, catalog number: 555274 , 553730 and 553032 , respectively)
- Concanavalin A (Sigma-Aldrich, catalog number: C5275 )
- 3H-thymidine (BARC)
- Trypan Blue (Sigma-Aldrich, catalog number: T8154 )
- RPMI-10 (see Recipes)
Equipment
- Haemocytometer
- Humidified CO2 incubator
- Laminar air flow bio-safety cabinet
- Centrifuge
- Gamma-irradiator
- Microscope
- Flow-cytometer
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kumar, P. and Bhaskar, S. (2016). Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction. Bio-protocol 6(5): e1750. DOI: 10.21769/BioProtoc.1750.
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分类
免疫学 > 免疫细胞功能 > 树突细胞
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