测量卵清蛋白（OVA)特异性CD8+T淋巴细胞杀伤力的体内实验

Nada Chaoul; Catherine Fayolle; Claude Leclerc

doi:10.21769/BioProtoc.1838

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Peer-reviewed

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

测量卵清蛋白（OVA)特异性CD8+T淋巴细胞杀伤力的体内实验

Nada Chaoul

Catherine Fayolle

Claude Leclerc email

发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1838 浏览次数: 22664

评审: Lee-Hwa TaiMartin V KolevAnonymous reviewer(s)

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Abstract

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 (IVK). Here we describe the protocol for the lysis of cells expressing a CD8⁺ T cell epitope of the OVA protein (SIINFEKL). Mice are previously immunized with the OVA protein and 7 days after immunization, they receive a mix of target cells, prepared from naive C57BL/6 spleen cells pulsed with the SIINFEKL peptide and labeled with high level of CFSE and of non-pulsed control cells labeled with low level of CFSE. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE^high cells and CFSE^low cells. The percentage of lysis is calculated by the difference between CFSE high versus low in immunized vs non-immunized mice.

Measuring the ability of antigen-specific CD8⁺ T cell 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 (细胞毒性T细胞)

CD8 T cells (CD8 T细胞)

Killing assay (杀法)

In vivo assay (体内试验)

Ovalbumin (卵清蛋白)

Materials and Reagents

0.5 ml insulin syringe (Terumo Corporation, catalog number: SS05M2713M )
Falcon^® 50 ml high clarity PP centrifuge tube (Corning, catalog number: 352070 )
40 μm nylon mesh cell strainer (Corning, catalog number: 352340 )
6-well plate (TPP Techno Plastic Products AG., catalog number: 92006 ) 
Cell counting material  
5 ml round bottom polystyrene test tube (Corning, catalog number: 352235 )
C57BL/6 mice
CpG B-1826 (5’-TCCATGACGTTCCTGACGTT-3’) (Sigma-Aldrich, PrOligo, custom made)  
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniumethyl sulfate (DOTAP) (Roche Diagnostics, catalog number: 11202375001 )
Albumin from chicken egg white (OVA protein) (Sigma-Aldrich, catalog number: A5503 )
Sterile DPBS, 1x (Life Technologies, Gibco, catalog number: 14190-094 )
Note: Currently, it is “DPBS, no calcium, no magnesium (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190-094 )”.
Carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, catalog number: C1157 )
Note: Currently, it is “(5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester) (CFSE) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: C1157)”.
ACK lysing buffer (1x) (Lonza Group Ltd., catalog number: 10548E )  
RPMI 1640 Medium, GlutaMAX^TM (Thermo Fisher Scientific, Gibco^TM, catalog number: 61870-010 )
Penicillin/streptomycin (Life Technologies, Gibco^TM, catalog number: 15140-12 )
Note: Currently, it is “Penicillin/streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )”.
Peptide SIINFEKL (PolyPeptide Group, custom made)
Fetal calf serum (Thermo Fischer Scientific, Hyclone^TM, catalog number: SV30160.03 )
Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
FACS buffer (see Recipes)

Equipment

Dissection kit (containing scissors, curved forceps)
Centrifuge (Eppendorf, model: 5810R )
MyBath^TM 4 mini water bath (Benchmark Scientific, model: B2000-4 )
Flow cytometer: Cyan (Beckman Coulter) or Fortessa (Becton Dickinson)

Procedure

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Chaoul, N., Fayolle, C. and Leclerc, C. (2016). In vivo OVA-specific Cytotoxic CD8⁺ T Cell Killing Assay. Bio-protocol 6(12): e1838. DOI: 10.21769/BioProtoc.1838.