Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions
流动状态下T淋巴细胞对肿瘤细胞的粘着强度分析
发布: 2013年10月20日第3卷第20期 DOI: 10.21769/BioProtoc.936 浏览次数: 10127
评审: Lin FangFanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
Abstract
This method allows evaluating the relative adhesion strength between T lymphocytes and specific adherent target cells using a shear force in flow chambers. It is based on the measure of the resistance of conjugates formed between T cells and adherent tumor cells to shear stress in a microfluidic system. For this purpose, T cells, stained with a CellTracker probe, are added into flow channels containing a monolayer of adherent target cells and their progressive detachment under a constant shear stress is then recorded using a fluorescent microscope.
Materials and Reagents
- Adherent tumor cells [such as non-small cell lung carcinoma (NSCLC) cell lines]
- Specific T-cell clones (generated either from autologous tumor-infiltrating T lymphocytes (TIL) or peripheral blood lymphocytes (PBL))
- RPMI 1640 (Life Technologies, Gibco®, catalog number: 61870044 )
- DMEM-F12 (Life Technologies, Gibco®, catalog number: 31331093 )
- UltroserG (Pall, catalog number: 15950-017 )
- Fetal Bovin Serum (Life Technologies, Gibco®, catalog number: 10270-106 )
- Human serum AB (Institut de Biotechnologies Jacques Boy)
- Penicillin and streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
- Sodium Pyruvate (Life Technologies, Gibco®, catalog number 11360029 )
- IL-2
- 10x PBS (Life Technologies, Gibco®, catalog number: 70011-036 )
- CellTracker probe (CellTrackerTM Green CMFDA) (Life Technologies, Invitrogen™, catalog number: C2925 )
- Complete DMEM: tumor cell culture medium (LC medium) (see Recipes)
- RPMI-based T-cell complete medium (see Recipes)
Equipment
- Microscope Zeiss LSM-510 (ZEISS) with a heated incubation chamber and CO2 supply
- Micro-Slides VI, ibiTreat (ibidi GmbH, catalog number: 80606 ), two silicon tubes (1.6 mm of inner diameter) with a plastic clip, two Elbow Luer connectors (ibidi GmbH, catalog number: 80646 )
- Syringe pump (high flow rate > 50 ml/min)
- 60 ml syringe (Becton, Dickinson and Company, catalog number: 300866 )
- Humidified incubator at 37 °C with 5% CO2
- A recipient for waste flow buffer (Erlenmeyer)
- Centrifuge (Beckman Coulter, model: GS-6R )
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Boutet, M., Franciszkiewicz, K., Floc’h, A. L. and Mami-Chouaib, F. (2013). Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions. Bio-protocol 3(20): e936. DOI: 10.21769/BioProtoc.936.
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分类
癌症生物学 > 通用技术 > 细胞生物学试验
细胞生物学 > 细胞成像 > 荧光
免疫学 > 免疫细胞功能 > 淋巴细胞
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