Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells
骨髓巨噬细胞和肿瘤细胞的跨室共培养
发布: 2014年05月05日第4卷第9期 DOI: 10.21769/BioProtoc.1121 浏览次数: 23053
评审: Lin FangFanglian HeHui Zhu
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参见作者原研究论文
The authors used this protocol in:
Mar 2013
Abstract
Bone is a primary site of metastasis from prostate and breast cancers. Bone marrow macrophages (BMMs) are mediators of inflammatory processes and are thought to promote tumor growth in the skeletal sites. In order to elucidate how their interactions with tumor cells impact aggressiveness of metastatic tumors in bone in vitro methods are required. By employing a system in which BMMs and tumor cells are grown separately, yet share the media and exchange soluble factors, contribution of each cell type in the context of BMM-tumor cell relationship in the bone marrow can be investigated. Additional advantages of this system include the ability to study: 1) phenotypic changes in BMMs and tumor cells upon co-culture; 2) cell-specific modulation of protein and gene expression; and 3) effects on proliferation and cell survival. It is noteworthy, that this transwell co-culture system is not limited to BMMs and tumor cells and can be easily modified to include other components of bone marrow microenvironment (e.g., adipocytes, stromal cells, osteoblasts).
Materials and Reagents
- Mice (FvBN strain)
- PC3 cells (ATCC, catalog number: CRL 7934 )
- L929 cells (ATCC, catalog number: VR-1404 )
- DMEM (Sigma-Aldrich, catalog number: D2902 )
- MEM-alpha (Sigma-Aldrich, catalog number: M0644 )
- Fetal bovine serum (Thermo Fisher Scientific, HyClone catalog number: SH3008803 )
- Pencillin-Streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
- 0.25% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25200-056 )
- 1x Sterile PBS (Sigma-Aldrich, catalog number: P3813 )
- 70% ethanol
- RNeasy Plus Mini Kit (QIAGEN, catalog number: 74134 )
- QIAshredder (QIAGEN, catalog number: 79654 )
- RLT buffer (part of RNeasy Plus Mini Kit, shown above), includes 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
- Sucrose (Sigma-Aldrich, catalog number: S9378 )
- MES (Sigma-Aldrich, catalog number: M8250 )
- Complete DMEM (see Recipes)
- L929 conditioned media (see Recipes)
- BMM media (see Recipes)
- SME lysis buffer (see Recipes)
Equipment
- 6-well plates (Corning, Costar®, catalog number: 3516 )
- Transwell inserts (Corning, catalog number: 3412 )
- Petri dishes (Thermo Fisher Scientific, catalog number: 08-757-13 )
- 100 mm tissue culture dishes (Corning, catalog number: 430293 )
- T75 flasks (Corning, catalog number: 430725 )
- Cell scrapers (SARSTEDT AG, catalog number: 83.1830 )
- Sterile fine-tip transfer pipets (Thermo Fisher Scientific, catalog number: 232-1S )
- 10 ml syringes (Luer-Lok) (BD, catalog number: 309604 )
- 5, 10, & 25 ml serological pipets
- Needles (26 Gauge & 20 Gauge) (BD, catalog numbers: 305111 & 305176 , respectively)
- Dissecting forceps and scissors
- Scalpel
- 1.5 ml tubes (Thermo Fisher Scientific, catalog number: 3464 )
- 15 ml centrifuge tubes (Thermo Fisher Scientific, Nunc®, catalog number: 339650 )
- 50 ml centrifuge tubes (Thermo Fisher Scientific, Nunc®, catalog number: 339652 )
- 5 K Millipore centrifugal concentrator (Amicon Ultra-4) (Millipore, model: UFC800596 )
- Centrifuge
- Incubator (5% CO2, 37 °C) (Napco Series 8000 DH CO2 Incubator)
- Biosafety cabinet (The Baker Company, SterilGARD®)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Herroon, M. K. and Podgorski, I. (2014). Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells. Bio-protocol 4(9): e1121. DOI: 10.21769/BioProtoc.1121.
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分类
癌症生物学 > 通用技术 > 细胞生物学试验
癌症生物学 > 肿瘤免疫学 > 肿瘤微环境
细胞生物学 > 组织分析 > 组织分离
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