In vitro Inflammasome Assay
体外炎性体试验
发布: 2014年06月05日第4卷第11期 DOI: 10.21769/BioProtoc.1142 浏览次数: 14099
评审: Lee-Hwa TaiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
Abstract
Innate immune cells sense pathogen and danger-associated molecular patterns (PAMPs and DAMPs) through a range of innate immune pattern recognition receptors (PRRs). One type of PRRs are the Nod-like receptors (NLRs), which form inflammasomes; a molecular platform required for the recruitment and activation of Caspase-1, which in turn cleaves and activates IL-1β, IL-18. Examples of inflammasome forming NLRs are NLRP3, NLRP1, NAIP and NLRC4. We can easily identify new inflammasome activators by performing the following protocol.
Materials and Reagents
- Mice (e.g. C57B/6)
- DMEM (Life Technologies, Gibco®, catalog number: 10566-024 )
- 1% Penicillin-Streptomycin (10,000 U/ml) (Life Technologies, Gibco®, catalog number: 15140148 )
- 1x PBS
- Accutase (PAA Laboratories GmbH, catalog number: L11-007 )
- Ultrapure Escherichia coli (E.coli) K12 LPS (Life Technologies, InvitrogenTM, catalog number: tlrl-peklps )
- IL-1β ELISA (eBioscience, catalog number: 88-7013-88 )
- Primary antibodies against IL-1β (e.g. R&D System, catalog number: AF-401-NA ) and Caspase-1 (e.g. Aidpogen International, catalog number: AG-20B-0042-C100 ) (suitable for western blot)
- HRP-conjugated secondary antibody (e.g. Cell Signaling anti-mouse HRP, Cell Signaling Technology, catalog number: 7076 )
- Nitrocellulose membrane (0.45 µm) (GE Healthcare, Hybond, catalog number: 95038-402 )
- 5% sodium azide in water (Sigma-Aldrich, catalog number: 26628-22-8 )
- Skim milk powder (Sigma-Aldrich, or your local grocer)
- ECL solution (Pierce, catalog number: 34095 or GE Healthcare, catalog number: RPN2133 )
- 1 M dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
- Stimulus: e.g. 1 M ATP (Sigma-Aldrich, catalog number: A26209 ), 10 mM nigericin (Sigma-Aldrich, catalog number: N7143 ), 300 µg/ml monosodium urate (Michigan State University, catalog number: U2875 )
- Flushing medium (see Recipes)
- Red blood cell lysis solution (see Recipes)
- Bone-marrow-derived macrophage (BMM) culture medium (see Recipes)
- Ponceau staining solution (see Recipes)
- 3x western blot sample buffer (see Recipes)
- Blocking buffer (see Recipes)
- Running buffer (see Recipes)
- Blotting buffer (see Recipes)
Equipment
- Razor blade
- Laminar flow hood
- Bench top centrifuge with 96-well plate adaptors
- 10 ml syringe
- 22 G gauge needles
- 18 G blunt needles
- 100-µm cells strainers
- 10 cm cell-culture treated petri dishes
- MaxiSorb ELISA plates (Nunc®)
- 96-well tissue culture plate
- ELISA plate reader
- Western blot equipment (protean mini 1 mm) (Bio-Rad Laboratories)
- Film, developer, dark room or equivalent development equipment
- Parafilm or plastic
Software
- ELISA analysis software
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:
- Zaiss, M. M. and Maslowski, K. M. (2014). In vitro Inflammasome Assay. Bio-protocol 4(11): e1142. DOI: 10.21769/BioProtoc.1142.
- Zaiss, M. M., Maslowski, K. M., Mosconi, I., Guenat, N., Marsland, B. J. and Harris, N. L. (2013). IL-1beta suppresses innate IL-25 and IL-33 production and maintains helminth chronicity. PLoS Pathog 9(8): e1003531.
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分类
免疫学 > 免疫细胞功能 > 细胞因子
生物化学 > 蛋白质 > 免疫检测 > 酶联免疫吸附试验（ELISA）
免疫学 > 免疫细胞分离 > 模式识别受体
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