In vivo Priming of T Cells with in vitro Pulsed Dendritic Cells: Popliteal Lymph Node Assay
使用体外脉冲树突状细胞体外致敏T细胞: 腘窝淋巴结实验
发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2531 浏览次数: 11919
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2017
Abstract
One-way mixed lymphocyte reaction (MLR) is a classic tool to measure how T cells react to external stimuli. However, MLR is an in vitro reaction system, which shows different response intensity compared with in vivo trails sometimes due to the lack of cytokines, tissue matrix and other immune response associated factors. The following popliteal lymph node assay (PLNA) protocol is designed to test the T cells antigen-specific reaction in vivo by using ovalbumin (OVA) specific reacted transgenic mouse OT-1 and OT-2.
Keywords: Mixed lymphocyte reaction (MLR) (混合淋巴细胞反应（MLR）)
Popliteal lymph node assay (PLNA) (腘窝淋巴结实验（PLNA）)
Antigen presenting cells (APC) (抗原呈递细胞（APC）)
Dendritic cells (DC) (树突状细胞（DC）)
Flow cytometry (流式细胞仪)
Ovalbumin antigen (卵清蛋白抗原)
OT-1 (OT-1)
OT-2 (OT-2)
Background
In transplantation, autoimmune and infection studies, one-way mixed lymphocyte reaction (MLR) is a helpful parameter to assess whether T-cell proliferation is increased or inhibited in response to antigen present cells (APC) or other external stimuli. This is a classic functional test, which demonstrates how the T-cell is affected by the test reagents, such as immune-suppress drug (Hou et al., 2015; Zhang et al., 2015), negative cell or exosome vaccines (Ma et al., 2016; Cai et al., 2017).
However, this system has some limits due to the fact that it is in vitro immune reaction system. In vivo immune reaction is affected by bio-microenvironment, such as cytokines, chemokines, hormones secretion, tissue matrix and other unknown bio-factors.
Herein, the popliteal lymph node assay (PLNA) was designed as an in vivo tool to test the increased or decreased immune response by using an ovalbumin (OVA) antigen-specific reaction system (Bhatt et al., 2014; Cai et al., 2017).
Materials and Reagents
- Gloves, mask and lab coat
- Pipette tips
10 μl (FUKAEKASEI and WATSON, catalog number: 110-207C )
200 μl (FUKAEKASEI and WATSON, catalog number: 110-705C )
1 ml (FUKAEKASEI and WATSON, catalog number: 110-706C ) - 27 G needle (Terumo Medical, catalog number: NN-2732R )
- 24-well tissue culture plate (Greiner Bio One International, CELLSTAR®, catalog number: 662160 )
- Nylon fiber column T (Wako Pure Chemical Industries, catalog number: 147-06721 )
- 21 G needle (Terumo Medical, catalog number: NN-2138R )
- Nylon mesh, 70 μm (AS ONE, catalog number: PA-70μ )
- Culture dish
3.5 ml (Greiner Bio One International, CELLSTAR®, catalog number: 627170 )
10 ml (Greiner Bio One International, CELLSTAR®, catalog number: 664160 ) - BD Falcon® disposable transfer pipets, 3 ml (Corning, Falcon®, catalog number: 357524 )
- 10 ml serological pipette (Greiner Bio One International, CELLSTAR®, catalog number: 607180 )
- Falcon® 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
- 1.5 ml round-bottom micro tube (FUKAEKASEI and WATSON, catalog number: 131-715C )
- Aluminum foil (Mitsubishi)
- C57BL6/Nslc (B6) mice
- 70% ethanol (Wako Pure Chemical Industries, catalog number: 324-00037 )
- Crashed ice and carrier
- Recombinant Murine GM-CSF (PeproTech, catalog number: 315-03 )
- Recombinant Murine IL-4 (PeproTech, catalog number: 214-14 )
- Recombinant Murine IFN-γ (PeproTech, catalog number: 315-05 )
- EndoGrade® Endotoxin-free ovalbumin (Hyglos, catalog number: 321000 )
- Distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230162 )
- Phosphate-buffered saline (PBS; 10x), pH 7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 70013073 )
- CellTraceTM Violet Cell Proliferation Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: C34557 )
- CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: C34554 )
- APC anti-mouse CD4 Antibody (BioLegend, catalog number: 100516 )
- APC Rat IgG2a, κ Isotype Ctrl Antibody (isotype) (BioLegend, catalog number: 400511 )
- PE anti-mouse CD8a Antibody (BioLegend, catalog number: 100708 )
- PE Rat IgG2a, κ Isotype Ctrl Antibody (isotype) (BioLegend, catalog number: 400507 )
- RPMI-1640 (Wako Pure Chemical Industries, catalog number: 189-02025 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
- 100x penicillin-streptomycin-glutamine (Thermo Fisher Scientific, catalog number: 10378016 )
- 100x MEM non-essential amino acids solution (Thermo Fisher Scientific, GibcoTM, catalog number: 11140 )
- 100 mM sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
- 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
- Complete medium (see Recipes)
Equipment
- Sanyo Bio clean bench (Panasonic Healthcare, catalog number: MCV-B131F )
- CO2 incubator (Panasonic Healthcare, catalog number: MCO-5ACUV-PJ )
- Autoclave (TOMY SEIKO, model: ES-315 )
- Tweezers (Weller, catalog number: 4SA )
- Centrifuge (Beckman Coulter, catalog number: Allegra X-12 )
- Water bath (Tokyo Rikakikai, EYELA, model: NTT-2000 )
- Dissecting instrument start set (NATSUME SEISAKUSHO, Nazme, catalog number: E-16-F )
- Flow cytometer (Beckman Coulter, model: Gallios )
Software
- FlowJo V.10.0.8. (FlowJo, LLC)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Cai, S., Fujino, M., Lu, L. and Li, X. (2017). In vivo Priming of T Cells with in vitro Pulsed Dendritic Cells: Popliteal Lymph Node Assay. Bio-protocol 7(17): e2531. DOI: 10.21769/BioProtoc.2531.
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分类
免疫学 > 动物模型 > 小鼠
细胞生物学 > 基于细胞的分析方法 > 胞外微环境
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