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Peer-reviewed

Efficient Isolation of Influenza Specific CTLs

流感特异性的杀伤性T细胞的高效分离

Prerak Trivedi Prerak Trivedi
Stuart I. Mannering Stuart I. Mannering

发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1847 浏览次数: 8637

评审: Jia LiAnonymous reviewer(s)

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Cover of The Journal of Experimental Medicine, featuring study using the protocol.

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Abstract

Human antigen-specific CD8+ T-cell clones are valuable tools for dissecting CD8+ T-cell responses against antigens derived from infectious agents, cancer and self antigens. Here we describe a protocol for isolating human antigen-specific CD8+ T cells. This protocol uses surface capture of IFNγ to identify antigen responsive cells that are then cloned by single-cell sorting. Here we use CD8+ T-cell responses to influenza matrix protein (MP) as an example, but this approach can be applied to any antigen specificity.

Keywords: Cloning (克隆) search CD8 (CD8) search CTL (CTL) search Human (人类) search T cell (T细胞) search

Materials and Reagents

  1. 50 ml Falcon tubes
  2. Greiner CELLSTAR®, 96-well round-bottom tissue culture plates (Sigma-Aldrich, catalog number: M9311 )
  3. Corning® Costar® 24 and 48 well tissue culture plates (Sigma-Aldrich, catalog number: CLS3524 and CLS3548 )
  4. 10 ml tube
  5. Vials
  6. Peripheral blood
  7. EBV-transformed B-cell lines (HLA-A2)
  8. Ficoll (GE Healthcare, catalog number: 17-1440-03 )
  9. Phosphate buffered saline (DPBS) (Lonza, catalog number: 17-512F )
  10. Trypan blue solution (0.4%) (Sigma-Aldrich, catalog number: T8154 )
  11. RPMI 1640 (Sigma-Aldrich, catalog number: R8758 )
  12. Synthetic peptide(s) (Purar Chemicals)
  13. Influenza matrix peptide 58-66 (MP58-66; GILGFVFTL) (GL-Biochem)
  14. 5% pooled human serum
  15. Cytokines IL-2 (Peprotech, catalog number: AF-200-02 ), IL-15 (Peprotech, catalog number: AF-200-15 )
  16. Human IFNγELISA Kit (BioLegend, catalog number: 430106 )
  17. Miltenyi IFN-γ secretion assay kit-PE (Miltenyi Biotec, catalog number: 130-054-202 )
  18. CD8-FITC (SK1) mAb (clone SK1) (Thermo Fisher Scientific, eBiosciencesTM, catalog number: 11-0087-42 )
  19. Fungizone (Thermo Fisher Scientific, catalog number: 15290-018 )
  20. Propidium iodide (Sigma-Aldrich, catalog number: 81845 )
  21. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
  22. Ionomycin (Sigma-Aldrich, catalog number: I0634 )
  23. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  24. HEPES (1 M) (Life technologies, GibcoTM, catalog number: 15630-080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080”.
  25. Non-essential amino acids (NEAA) (Life Technologies, GibcoTM, catalog number: 11140-050 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050”.
  26. GlutaMAXTM (Life Technologies, GibcoTM, catalog number: 35050-061 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061”.
  27. UltraPureTM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575-020 )
  28. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P0781 )
  29. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  30. Stock concentration of cytokines and peptides (see Recipes)
  31. Cell culture media (see Recipes)
  32. Staining buffer (see Recipes)
  33. Stock concentration of propidium iodide (PI) (see Recipes)
  34. Stock concentration of PMA and ionomycin (see Recipes)
  35. Stock concentration of fungizone (see Recipes)

Equipment

  1. Megafuge 40R centrifuge (ThermoFisher, catalog number: 75004503 )
  2. Neubauer chamber
  3. Freezing container, Nalgene® Mr. Frosty (Sigma-Aldrich, C1562 )
  4. Cell culture CO2 incubator (Panasonic Healthcare Co., model: MCO-20AIC ) with 5% CO2
  5. Flow cytometer (BD Biosciences, model: BD FACSAria III )
  6. Liquid nitrogen dewar

Procedure

English
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文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Trivedi, P. and Mannering, S. I. (2016). Efficient Isolation of Influenza Specific CTLs. Bio-protocol 6(12): e1847. DOI: 10.21769/BioProtoc.1847.

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分类

免疫学 > 免疫细胞功能 > 抗原特异反应

细胞生物学 > 细胞分离和培养 > 细胞分离

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