Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages
庆大霉素保护试验测定细菌在巨噬细胞中的存活能力
发布: 2014年09月20日第4卷第18期 DOI: 10.21769/BioProtoc.1235 浏览次数: 22745
评审: Kanika GeraAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
Abstract
Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 cells, a widely used macrophage cell line. Bacterial mutants defective in intracellular survival within macrophages can be delineated using this assay.
Materials and Reagents
- RAW 264.7 cells (ATCC, TIB-71 )
- E. coli CFT073 or other strains of bacteria
- LB broth (Beckton Dickinson, catalog number: 244610 )
- Agar (Beckton Dickinson, catalog number: 214010 )
- RPMI1640 (Life Technologies, catalog number: 11875-093 )
- Gentamicin 50 mg/ml (Life Technologies, catalog number: 15750-102 )
- PBS (pH 7.4)
- LB plates (see Recipes)
- Certified, heat inactivated fetal bovine serum (FBS) (Life Technologies, catalog number: 10082139 ) (see Recipes)
- 100x penicillin, streptomycin and glutamine (PSG) (Life Technologies, catalog number: 10378-016 ) (see Recipes)
- Saponin (Sigma-Aldrich, catalog number: 8047-15-2) (see Recipes)
Equipment
- 24 well plates (Corning, Costar®, catalog number: 3524 )
- 0.22 micron filter (Millipore, catalog number: SCGP00525 )
- 37 °C, 5% CO2 incubator
- Sorvall Legend RT table top centrifuge
- Shimadzu UV-1601PC Spectrophotometer
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Subashchandrabose, S. and Mobley, H. L. (2014). Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages. Bio-protocol 4(18): e1235. DOI: 10.21769/BioProtoc.1235.
- Subashchandrabose, S., Smith, S. N., Spurbeck, R. R., Kole, M. M. and Mobley, H. L. (2013). Genome-wide detection of fitness genes in uropathogenic Escherichia coli during systemic infection. PLoS Pathog 9(12): e1003788.
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分类
微生物学 > 抗微生物试验 > 杀伤试验
微生物学 > 微生物-宿主相互作用 > 体外实验模型
免疫学 > 宿主防御 > 综合
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