鼠源记忆性T淋巴细胞的体外分化

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

doi:10.21769/BioProtoc.1171

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Peer-reviewed

Murine in vitro Memory T Cell Differentiation

鼠源记忆性T淋巴细胞的体外分化

MK Myoungjoo V. Kim email

发布: 2014年07月05日第4卷第13期 DOI: 10.21769/BioProtoc.1171 浏览次数: 14541

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

Cell Imaging - A Special Collection for Cell Bio 2023

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Abstract

Upon pathogen encounter, naïve CD8⁺ T cells are primed and undergo massive clonal expansion. A fraction of effector CD8⁺ T cells remains during the contraction phase and differentiate into memory T cells critical for mounting robust recall responses in response to secondary infection. Low frequency of memory T cells in vivo is a major obstacle to investigate their functional aspects including migration capacity and genetic regulation. Here, we describe detailed protocol for memory T cell differentiation developed by von Andrian’s group to generate large number of CD44^hiCD62L^hi antigen-specific memory T cells in vitro.

Keywords: Ag-specific memory CD8 T cell (记忆CD8 T细胞抗原特异性)

OT-I (催产素- I)

IL-15 (白细胞介素15)

In vitro (在体外)

CD44+CD62L+ central memory T cell (CD44 + CD62L +中央记忆T细胞)

Materials and Reagents

Recombinant mouse IL-15 (rmIL15) (BioLegend, catalog number: 566302 )
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 )
100x 1 M Hepes (Life Technologies, Gibco^®, catalog number: 15630-080 )
100x MEM non-essential amino acids (Life Technologies, Gibco^®, catalog number: 11140-050 )
100x sodium pyruvate (100 mM) (Life Technologies, Gibco^®, catalog number: 11360-070 )
100x 2-mercaptoethanol (Life Technologies, Gibco^®, catalog number: 21985-023 )
OVA_257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951 )
Ficoll-Paque^TM Premium 1.084 (GE Healthcare, catalog number: 17-5446-02 )
Antibodies:
1. Anti-CD44 PerCpCy5.5 (clone: IM7) (eBioscience, catalog number: 45-0441 )
2. Anti-CD62L APC (clone: MEL-14) (eBioscience, catalog number: 17-0621 )
RBC lysis buffer (eBioscience, catalog number: 00-4333-57 )
Bovine serum albumin (Thermo Fisher Scientific, catalog number: BP1605-100 )
NaN₃ (Sigma-Aldrich, catalog number: S8032 )
T cell media (see Recipes)
Staining buffer (in PBS) (see Recipes)

Equipment

Centrifuge (Thermo Fischer Scientific, Sorvall^TM Legend RT)
70 µm cell strainer (BD Biosciences, Falcon^®, catalog number: 352350 )
15 ml and 50ml Falcon tubes
24 well plates (BD Biosciences, Falcon^®, catalog number: 353226 )
T75 culture flask (Corning, catalog number: 430641 )
37 °C, 5% CO₂ cell culture incubator

Procedure

English

中文翻译

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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 vitro Memory T Cell Differentiation. Bio-protocol 4(13): e1171. DOI: 10.21769/BioProtoc.1171.