流式细胞法体外评估免疫突触的形成

Bo-Ra Na; Chang-Duk Jun

doi:10.21769/BioProtoc.1758

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Peer-reviewed

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

Bo-Ra Na

Chang-Duk Jun email

发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1758 浏览次数: 16486

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:

Cover of The Journal of Cell Biology, featuring study using the protocol.

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
Falcon^TM Cell strainer (40 μm Nylon) (Corning, catalog number: 352340 )
1 ml syringe
Falcon^TM 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 Probes^TM, catalog number: C7025”.
CellTracker Orange CMRA (Life Technologies, Molecular Probes^®, catalog number: C34551 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, 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, Gibco^TM, catalog number: 31800 )
10% heat-inactivated fetal bovine serum (FBS) (Siyaku, Wako Pure Chemical Industries, AusGeneX, catalog number: FBSUS500 )
Penicillin/streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140 )
Non-essential amino acids (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140 )
Sodium pyruvate (Thermo Fisher Scientific, Gibco^TM, 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

English

中文翻译

文章信息

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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.