Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation
采用Percoll密度梯度离心法分离小鼠肿瘤浸润性白细胞
发布: 2013年09月05日第3卷第17期 DOI: 10.21769/BioProtoc.892 浏览次数: 43698
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
Abstract
A hallmark of cancer-associated inflammation is the infiltration of leukocytes into tumors, which is believed to be recruited by chemokines. Some infiltrating leukocytes such as macrophages often promote tumor growth by producing growth-inducing and angiogenic factors. Here, we described a method of isolating tumor-infiltrating leukocytes with Percoll density gradient, because Percoll possesses a low viscosity, a low osmolarity and no toxicity to cells. Different leukocyte populations are isolated based on their density and collected at the interface between 40% and 80% discontinuous Percoll gradient.
Materials and Reagents
- Mice with tumors
- Collagenase, type IV (Sigma-Aldrich, catalog number: C5138 )
- DNase, type IV (Sigma-Aldrich, catalog number: D5025 )
- Fungizone Antimycotic (Life Technologies, InvitrogenTM, catalog number: 15290-026 )
- Hank's Balanced Salt Solution (HBSS), without Calcium Chloride or Magnesium Chloride (Lonza, catalog number: 10-547F )
- Hyaluronidase, Type V (Sigma-Aldrich, catalog number: H6254 )
- Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A4503 )
- 0.5 M EDTA (Quality Biological Inc, catalog number: qb351-027-721 )
- Percoll (GE Healthcare, catalog number: 17-0891-01 )
- Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Lonza, catalog number: 12-614F )
- Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: SH30070-03 )
- 10x PBS (Life Technologies, Gibco®, catalog number: 70011 )
- ACK lysing buffer (Lonza, catalog number: 10-548E )
- 10x Triple Enzyme Stock Solution (see Recipes)
- Wash buffer (see Recipes)
- 40% Percoll (see Recipes)
- 80% Percoll (see Recipes)
Equipment
- 25 ml canted Neck and Blue Plug Seal Cap, non-vented tissue culture flasks (BD Biosciences, Falcon®, catalog number: 353018 )
- Shaker (Lab-Line, model: 1314 )
- Centrifuge
- 100 mm Petri dish
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Liu, Y., Chen, K., Wang, C., Gong, W., Yoshimura, T., Wang, J. M. and Liu, M. (2013). Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation. Bio-protocol 3(17): e892. DOI: 10.21769/BioProtoc.892.
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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 > 生物化学试验
细胞生物学 > 细胞分离和培养 > 细胞分离
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