In vitro Assessment of Immunological Synapse Formation by Flow Cytometry
流式细胞法体外评估免疫突触的形成
发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1758 浏览次数: 17664
评审: Ralph BottcherMartin V KolevAlka Mehra
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参见作者原研究论文
The authors used this protocol in:
Apr 2015
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Abstract
In adaptive immune system, formation of immunological synapse between T cells and antigen presenting cells (dendritic cells, B cells, and macrophages) or target cells (tumor cells and viral-infected cells) is critical for the execution of T cell immune responses via cytokine secretion or direct killing activity. Here, we describe the practical methods that directly measure the number of conjugates as a result of immunological synapse formation between T cells and superantigen-loaded B cells or between cytotoxic T cells and antigen-loaded target cells by dual-color flow cytometry.
Keywords: T cells (T细胞)
Antigen-presenting cells (抗原提呈细胞)
Immunological synapse (免疫突触)
Adhesion (粘附)
Conjugation (共轭)
Materials and Reagents
- 15 ml and 50 ml conical tube
- 12 well cell culture plate
- FalconTM Cell strainer (40 μm Nylon) (Corning, catalog number: 352340 )
- 1 ml syringe
- FalconTM 5 ml Polystyrene Round-Bottom tube (Corning, catalog number: 352052 )
- MACS MS column (Miltenyi Biotec, catalog number: 130-042-201 ) and MACS separator (Miltenyi Biotec)
- Jurkat T cell (ATCC, catalog number: TIB-152 )
- Raji B cell (ATCC, catalog number: CCL-86 )
- EL4 cell (ATCC, catalog number: TIB-39 )
- C57BL/6 mice (8-10 weeks)
- OTI mice (8-10 weeks) (THE JACKSON LABORATORY, catalog number: 021773 )
Note: These mice have the transgenic T cell receptor designed to recognize ovalbumin residues 257-264 in the context of H2Kb. - Staphylococcus Enterotoxin E (SEE) (Toxin Technology, catalog number: ET404 )
- Staphylococcus Enterotoxin B (SEB) (Toxin Technology, catalog number: BT202 )
- OVA 257–264 peptide (InvivoGen, catalog number: vac-sin )
- CellTracker Green CMFDA (Life Technologies, Molecular Probes®, catalog number: C7025 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C7025”. - CellTracker Orange CMRA (Life Technologies, Molecular Probes®, catalog number: C34551 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C34551”. - RBC lysis buffer (BioLegend, catalog number: 42030 )
- Mouse CD3+ T cell enrichment column kit (R&D Systems, catalog number: MTCC535 )
- Mouse B cell enrichment kit (STEMCELL Technologies, catalog number: 19754A )
- CD8a (Ly-2) MicroBeads (Miltenyi Biotec, catalog number: 130-049-401 )
- Recombinant human IL-2 (rhIL-2) (NIH AIDS Reagent Program, catalog number: 11697 )
- RPMI1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 31800 )
- 10% heat-inactivated fetal bovine serum (FBS) (Siyaku, Wako Pure Chemical Industries, AusGeneX, catalog number: FBSUS500 )
- Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
- Non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140 )
- Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360 )
- 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M-7522 )
- Complete RPMI1640 medium (see Recipes)
- Mouse T cell medium (see Recipes)
Equipment
- Water Jacketed CO2 incubator (37 °C, 5% CO2) (Thermo Fisher Scientific)
- FACSCanto instrument (BD Bioscience)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Na, B. and Jun, C. (2016). In vitro Assessment of Immunological Synapse Formation by Flow Cytometry. Bio-protocol 6(6): e1758. DOI: 10.21769/BioProtoc.1758.
- Na, B. R., Kim, H. R., Piragyte, I., Oh, H. M., Kwon, M. S., Akber, U., Lee, H. S., Park, D. S., Song, W. K., Park, Z. Y., Im, S. H., Rho, M. C., Hyun, Y. M., Kim, M. and Jun, C. D. (2015). TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse. J Cell Biol 209(1): 143-162.
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分类
免疫学 > 免疫细胞染色 > 流式细胞术
免疫学 > 免疫细胞功能 > 淋巴细胞
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