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In vitro T Cell–DC and T Cell–T Cell Clustering Assays

T淋巴细胞与树突细胞以及T淋巴细胞之间的体外簇集试验

AG Audrey Gérard

发布: 2013年10月20日第3卷第20期 DOI: 10.21769/BioProtoc.933 浏览次数: 15737

评审: Anonymous reviewer(s)

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Apr 2013

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Abstract

To get activated, T cells need to find their cognate antigen at the surface of an antigen-presenting cell (APC). Recognition of cognate antigen in the context of the MHC (Major histocompatibility complex) by the TCR (T-Cell Receptor) results in long lasting interactions between T cells and APCs. Subsequently, T cells form homotypic interactions with each other, which is seen as a hallmark of T cell activation. This protocol describes a method to analyze T-APC and T-T conjugation.

Materials and Reagents

  1. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-093 )
  2. FBS (Hyclone, catalog number: SH30396-03 )
  3. Penicillin-Streptomycin-Glutamine Solution (Life Technologies, Gibco®, catalog number: 10378-016 )
  4. 2-mercaptoethanol (Life Technologies, Invitrogen™, catalog number: 21985-023 )
  5. Phorbol 12-myristate-13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
  6. Ionomycin (Sigma-Aldrich, catalog number: 19657 )
  7. 5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester (5(6)-CFDA, SE) (CFSE) (Life Technologies, Molecular Probes®, catalog number: C1157 )
  8. 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one (DDAO) (Life Technologies, Invitrogen™, catalog number: C34553 )
  9. 5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino) Tetramethylrhodamine (CMTMR) (Life Technologies, Invitrogen™, catalog number: C2927 )
  10. 16% Paraformaldehyde Solution (PFA) (16% Formaldehyde, EM Grade) (Electron Microscopy Sciences, catalog number: 15710 )
  11. Vectashield Hardset with Dapi (VWR International, catalog number: 101098-050 )
  12. Poly-L-Lysine Solution 0.1% (w/v) in H2O (Sigma-Aldrich, catalog number: P8920 )
  13. SL8 (Ovalbumin (257-264)) (AnaSpec, catalog number: 60193 )
  14. PBS (Life Technologies, catalog number: 14190-44 )
  15. Media (see Recipes)
  16. 2% PFA (see Recipes)
  17. Poly-L-Lysine-coated chamber (see Recipes)

Equipment

  1. Centrifuge
  2. Flow Cytometer
  3. Epifluorescent microscope
  4. 37 °C, 5% CO2 Cell culture incubator
  5. 24 well plate (Corning, Costar®, catalog number: 3524 )
  6. 40 μm Cell strainer (BD Biosciences, Falcon®, catalog number: 352340 )
  7. FACS tube, polystyrene (BD Biosciences, Falcon®, catalog number: 352054 )
  8. 8-well chamber slide (Thermo Fisher Scientific, Lab-TekTM, catalog number: 177445 )

Procedure

English
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文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Gérard, A. (2013). In vitro T Cell–DC and T Cell–T Cell Clustering Assays. Bio-protocol 3(20): e933. DOI: 10.21769/BioProtoc.933.

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分类

免疫学 > 免疫细胞功能 > 淋巴细胞

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