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In-vivo gp100-specific Cytotoxic CD8+ T Cell Killing Assay

体内gp100特异的细胞毒CD8+ T 细胞杀伤实验

Mahdia Benkhoucha Mahdia Benkhoucha
NM Nicolas Molnarfi
EB Elodie Belnoue
MD Madiha Derouazi
PL Patrice H Lalive

发布: 2018年11月20日第8卷第22期 DOI: 10.21769/BioProtoc.3082 浏览次数: 8756

评审: Alka MehraKathrin SutterAnonymous reviewer(s)

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Abstract

Cytotoxic CD8+ T lymphocytes (CTLs) represent a crucial component of the adaptive immune system and play a prominent role in the anti-tumor immune responses of both mice and humans.

Cytotoxic CD8+ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8+ T cell killing activity, we use the in vivo killing assay (IVKA). Here, we describe the protocol for the lysis of cells expressing a CD8+ T cell melanoma epitope of the hgp10025-33 protein (KVPRNQDWL). C57BL/6 recipient mice, receive first target cells, prepared from naive congenic (CD45.1) C57BL/6 spleen cells pulsed with the hgp10025-33 peptide and labeled with CFSE and of non-pulsed control cells labeled with Brilliant violet. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE cells and Brillant Violet (BV) cells. The percentage of lysis is calculated by the difference between CFSE versus BV.

Measuring the ability of antigen-specific CD8+ T cells to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.

Keywords: Cytotoxic T cells (细胞毒T细胞) search CD8 T cells (CD8 T细胞) search In vivo killing assay (体内杀伤实验) search Antigen specific CTL (抗原特异性CTL) search Tumor immunity (肿瘤免疫) search Cancer (癌症) search

Background

The advantage to use the two types of dye (CFSE and BV) is for their high stability in vivo, both dyes could be detectable even more than one week in vivo (based on our lab experience), on the other hand, as we used Galios cytometer, CFSE and BV are read respectively in fl1 vs. fl9 (fl1 and fl9 are two different lasers used by the FACS machine) which is very practical for the settings and compensations.

Materials and Reagents

  1. Tips
    0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440)
    1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502)
    101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509)
  2. 0.01-1 ml syringe (OnnifixR-F, catalog number: 9161406V)
  3. Falcon® 50 ml tubes (BD Biosciences, Falcon®, catalog number: 352098)
  4. Falcon® 15 ml tubes (BD Biosciences, Falcon®, catalog number: 352096)
  5. 40 μm Nylon mesh cell strainer (Corning, catalog number:352350)
  6. 70 μm Nylon cell strainer (Falcon, BD Biosciences)
  7. 150-cm2 flasks (TPP Techno Plastic Products, catalog number: 93150)
  8. 75 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353110)
  9. 6-well tissue culture plates (Corning, Falcon®, catalog number: 353224)
  10. 5 ml round bottom polystyrene test tube (Corning, Falcon®, catalog number: 352052)
  11. Female C57BL/6 mice (Janvier, France)
  12. Thy1.1+ Pmel-1 T-cell receptor (TCR) transgenic mice on a C57BL/6 CD45.2 background (Jackson Laboratory)
  13. hgp10025-33 (KVPRNQDWL) (ANASPEC, catalog number: AS-62589)
  14. Sterile DPBS, 1x (Life Technologies, Gibco, catalog number: 14190-094)
  15. Cell trace violet dye (BV) (Life Technologies, catalog number: 65-0842-85)
  16. IL-2 (50 IU/ml; PeproTech)
  17. RPMI 1640 Medium, GlutaMAXTM (Sigma-Aldrich, catalog number: R8758-500)
  18. Trypan blue (Gibco, catalog number: 15250061)
  19. HBSS (Bioconcept, catalog number: 3-02K32-I)
  20. Carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, catalog number: C34554)
  21. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl) 
  22. Bovine serum albumin (Sigma-Aldrich, catalog number: 12133C)
  23. PBS
  24. HEPES (Gibco, catalog number: 15630056)
  25. RBC lysis buffer (homemade) (one volume of Tris base 200 mM pH 7.5 and nine volumes of NH4Cl 150 mM) 
  26. DMEM (Gibco, catalog number: 12634010)
  27. Complete RPMI-1640 medium (see Recipes)
  28. FACS buffer (see Recipes)

Equipment

  1. Cell counting material (Automated cell counter or manual counter) 
  2. Pipetman® L (Gilson)
  3. Dissection kit (containing scissors, curved forceps)
  4. Hemocytometer
  5. Centrifuge
  6. MyBathTM water bath (Benchmark Scientific)
  7. Incubator 37 °C, 5% CO2
  8. Flow cytometer Galios (Beckman Coulter), ARIA FACS sorter (BD Bioscience)

Software

  1. FlowJo_V10
  2. GraphPad Prism software for PC, version 7.03, used for data analysis

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Benkhoucha, M., Molnarfi, N., Belnoue, E., Derouazi, M. and Lalive, P. H. (2018). In-vivo gp100-specific Cytotoxic CD8+ T Cell Killing Assay. Bio-protocol 8(22): e3082. DOI: 10.21769/BioProtoc.3082.

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分类

细胞生物学 > 基于细胞的分析方法 > 流式细胞术

癌症生物学 > 肿瘤免疫学 > 动物模型

免疫学 > 动物模型 > 小鼠

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