鼠体内 CD8+ T淋巴细胞杀菌分析

Myoungjoo V. Kim; Weiming  Ouyang; Will  Liao; Michael  Q.  Zhang; Ming  O.  Li

doi:10.21769/BioProtoc.1172

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Peer-reviewed

Murine in vivo CD8⁺ T Cell Killing Assay

鼠体内 CD8+ T淋巴细胞杀菌分析

MK Myoungjoo V. Kim email

发布: 2014年07月05日第4卷第13期 DOI: 10.21769/BioProtoc.1172 浏览次数: 19615

评审: Omar AkilAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Immunity, featuring study using the protocol.

Aug 2013

实验方案合集

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Abstract

Antigen-specific killing ability of effector CD8⁺ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8⁺ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8⁺ T cell responses. Effector CD8⁺ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into the previously immunized mice. Difference in CFSE expression levels clearly marks two distinct populations: Antigen-pulsed target cells-CFSE^low vs. unpulsed target cells-CFSE^hi. The frequencies between antigen-pulsed target cells and control target cells are used as readouts of antigen-specific killing.

Materials and Reagents

Splenocytes from a wild type mouse
PBS (Thermo Fisher Scientific, catalog number: BP399-20 )
Note: 10x solution, diluted to 1x in house in distilled water and sterilized by autoclave.
RBC lysis buffer (eBioscience, catalog number: 00-4333-57 )
HBSS without Ca²⁺ and Mg²⁺ (Life Technologies, Gibco^®, catalog number: 14175-095 )
RPMI-1640 medium (Life Technologies, Gibco^®, catalog number: 11875-119 )
Fetal bovine Serum (Atlanta Biologicals, catalog number: S11055H )
Penicillin/streptomycin (Gemini Bio-Products, catalog number: F52M00E )
L-Glutamine (Life Technologies, Gibco^®, catalog number: 25030-081 )
Trypan blue solution (Life Technologies, Gibco^®, catalog number: 15250-061 )
OVA_257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951 )
5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich, catalog number: 21888 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D-8418 )
Collagenase D (Sigma-Aldrich, catalog number: C-5138 )
Percoll (Sigma-Aldrich, catalog number: P-1644 )
Complete RPMI-1640 media (see Recipes)
100% percoll solution (see Recipes)

Equipment

Centrifuge (Thermo Fischer Scientific, Sorvall^TM Legend RT )
37 °C water bath
Hemocytometer
15 ml and 50 ml Falcon tubes
6 well plates (USA Scientific, CytoOne^®, catalog number: CC7682-7506 )
BD LSRII Flow Cytometer (BD)
70 µm cell strainer (BD Biosciences, Falcon^®, catalog number: 352350 )
5 ml polystyrene round-bottom tubes with cell-strainer cap (BD Biosciences, Falcon^®, catalog number: 352235 )
3 ml syringe (BD, catalog number: 14-823-435 )

Procedure

English

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如何引用

Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. and Li, M. O. (2014). Murine in vivo CD8⁺ T Cell Killing Assay. Bio-protocol 4(13): e1172. DOI: 10.21769/BioProtoc.1172.