Macrophage Phagocytosis Assay of Staphylococcus aureus by Flow Cytometry
通过流式细胞术进行葡萄球菌属巨噬细胞吞噬作用的测量
发布: 2015年02月20日第5卷第4期 DOI: 10.21769/BioProtoc.1406 浏览次数: 15251
评审: Fanglian HeAlka MehraAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
Abstract
This protocol describes a straightforward technique to evaluate the phagocytotic capacity of murine macrophages for Staphylococcus aureus (S. aureus). By staining S. aureus with Hexidium Iodide and staining murine bone marrow-derived macrophages (BMDMs) with FITC, the macrophage bacterial up-taking ability can be rapidly analyzed by flow cytometry. S. aureus is a Gram-positive bacteria causing severe human and animal infections. Host immune cells such as macrophages serve to eliminate S. aureus by phagocytosing the pathogen and save the host from life-threatening diseases. Study of host macrophage ability to phagocytose S. aureus is important for understanding the host-pathogen interaction and can help to elucidate the pathogenesis of S. aureus infection. This protocol may also be applied for macrophage phagocytotic assay of other gram-positive bacteria.
Materials and Reagents
- Cell line-L929 (ATCC, catalog number: CCL-1 )
- Staphylococcus aureus strain-Sanger 476 (Wellcome Trust Sanger Institute, catalog number: MSSA476 )
- Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco®, catalog number: 11965-092 )
- L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
- Penicillin-Streptomycin (Life Technologies, Gibco®, catalog number: 15140-148 )
- Fetal bovine serum (Hyclone, catalog number: SH30070.03 )
- DPBS (Life Technologies, Gibco®, catalog number: 14190 )
- EDTA (Sigma-Aldrich, catalog number: E6758 )
- Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25200-056 )
- ACK lysis buffer (Life Technologies, Gibco®, catalog number: A10492-01 )
- Tissue-culture water (Hyclone, catalog number: SH3052902 )
- Tryptic Soy Broth powder (TSB) (BD, catalog number: 211768 )
- Hexidium Iodide (Life Technologies, InvitrogenTM, catalog number: H7593 )
- FITC-anti-mouse-F4/80 (BioLegend, catalog number: 123107 )
- DMEM complete media with 10% FBS (D10) (see Recipes)
- Supernatant of L-cell cultural media (L-sup) (see Recipes)
- Bone marrow macrophage medium (BMM medium) (see Recipes)
- 500 mM-EDTA (see Recipes)
- 5 mM-EDTA (see Recipes)
- TSB (see Recipes)
- Hexidium Iodide stock (see Recipes)
Equipment
- 26G needle (Sigma-Aldrich, catalog number: Z192392-100 EA)
- Syringe (Terumo Medical Corporation, catalog number: SS-30L )
- Cell culture dish (Corning, catalog number: 430293 )
- 6-Well Plate (Falcon, catalog number: 351146 )
- 75 cm flask (Corning, catalog number: 430641 )
- Vacuum filtration system (Corning, catalog number: 430758 )
- 50 ml Corning tube (Corning, catalog number: 430290 )
- 5 ml flow cytometry tube (BD, catalog number: 352054 )
- Bacterial incubator shaker
- CO2 incubator
- Sorvall RT7 centrifuge
- Flow cytometer (FACSCanto)
Software
- FlowJo
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yan, Q., Ahn, S. H. and Fowler, V. G. (2015). Macrophage Phagocytosis Assay of Staphylococcus aureus by Flow Cytometry. Bio-protocol 5(4): e1406. DOI: 10.21769/BioProtoc.1406.
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分类
免疫学 > 免疫细胞功能 > 巨噬细胞
微生物学 > 微生物-宿主相互作用 > 细菌
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