Thioglycollate-elicited Peritoneal Macrophages Preparation and Arginase Activity Measurement in IL-4 Stimulated Macrophages
制备巯基乙酸盐诱导的腹水巨噬细胞并测量IL-4刺激后的精氨酸酶活性
发布: 2015年09月05日第5卷第17期 DOI: 10.21769/BioProtoc.1585 浏览次数: 22014
评审: Ivan ZanoniSabine Le SauxAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2015
Abstract
Macrophages are an essential cell population of innate immunity that plays important roles in inflammatory processes. Two main different phenotypes have been described with opposing activities: The classically activated macrophages (M1) and the alternatively activated macrophages (M2). Alternative activation of mouse macrophages can be induced by type 2 cytokines such as IL-4 and it is characterized by the regulation of the L-arginine metabolism. M2 macrophages convert arginine to ornithine and urea through the action of Arginase-1. Here we described a method for the isolation of peritoneal macrophages from thioglycollate-elicited mice and alternative activation by stimulation with IL-4. Intraperitoneal injection of thioglycollate elicits large numbers of macrophages into peritoneal cavity.
Keywords: Macrophages (巨噬细胞)
Thioglycollate (巯基乙酸)
Arginase (精)
IL-4 (IL-4)
Materials and Reagents
- Mice C57BL/6 male (age 8-12 weeks)
- Difco Fluid Thioglycollate medium (BD, catalog number: 225650 )
- 70% and 100% ethanol
- PBS (Lonza, catalog number: BE 17-515Q )
- RPMI 1640 (Lonza, catalog number: BE 12-115F )
- FBS (endotoxin<10 EU/ml) (Hyclone, catalog number: SV30160.03 )
- Penicillin-Streptomycin (Lonza, catalog number: BE 17-602E )
- Murine IL-4 (Peprotech, catalog number: 214-14 )
- Tris (Panreac Applichem, catalog number: 131940.1211 )
- NaCl (Merck KGaA, catalog number: 1.06404.1000 )
- EDTA (Sigma-Aldrich, catalog number: ED255 )
- Triton X-100 (Sigma-Aldrich, catalog number: 8787 )
- Protease inhibitor mixture (Sigma-Aldrich, catalog number: P8340 )
- Urea (Sigma-Aldrich, catalog number: 4883 )
- MnCl2 (Sigma-Aldrich, catalog number: 244589 )
- L-arginine monohydrochloride (Sigma-Aldrich, catalog number: A6969 )
- H2SO4 (Sigma-Aldrich, catalog number: 320501 )
- H3PO4 (Sigma-Aldrich, catalog number: 345245 )
- Isonitrosopropiophenone (Sigma-Aldrich, catalog number: I3502 )
- 12-well plates
- 1.5 ml Eppendorf tubes
- 50 ml sterile Falcon tubes
- Cell strainer (Becton Dickinson, catalog number: 352340 )
- 10 ml syringe
- 21G Needles
- 25G needles
- Lysis buffer (see Recipes)
- Heat-inactivated FBS (see Recipes)
- Stopping solution (see Recipes)
Equipment
- Bench-top refrigerated centrifuge
- Scissors
- Incubator (37 °C, 5% CO2 and 95 % humidity)
- Tissue culture hood (biosafety cabinet)
- Optical microscopy
- Neubauer cell counting chamber
- Espectrophotometer plate reader
- Thermoblock (Eppendorf Thermomixer Compact) or water bath
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jiménez-García, L., Herránz, S., Luque, A. and Hortelano, S. (2015). Thioglycollate-elicited Peritoneal Macrophages Preparation and Arginase Activity Measurement in IL-4 Stimulated Macrophages. Bio-protocol 5(17): e1585. DOI: 10.21769/BioProtoc.1585.
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分类
免疫学 > 免疫细胞功能 > 巨噬细胞
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