Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression
Bal液体调控成纤维细胞α-SMA表达的评估实验
发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2009 浏览次数: 9212
评审: Ivan ZanoniAchille BroggiBenoit Stijlemans
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The authors used this protocol in:
Mar 2016
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Abstract
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).
Background
Alveolar macrophages play an integral role in pulmonary fibrosis development by increasing the expression of TGF-β1 (He et al., 2011). Our prior data demonstrate that alveolar macrophages are a critical source of TGF-β1 as mice harboring a conditional deletion of TGF-β1 in macrophages were protected from pulmonary fibrosis (Larson-Casey et al., 2016). The expression of α-SMA is a defining feature of myofibroblasts, and TGF-β1 is a well-characterized pro-fibrotic mediator that induces transformation of fibroblasts to myofibroblasts both in vitro (Desmoulière et al., 1993) and in vivo (Sime et al., 1997). Prior studies exposed fibroblasts to recombinant TGF-β1 to show its effect on differentiation and function (Horowitz et al., 2007). Here we have developed a protocol for determining the ability of mouse BAL fluid to alter the differentiation of human lung fibroblasts to myofibroblasts, the cells that produce extracellular matrix proteins.
Materials and Reagents
- 6-well cell culture plates (Corning, Costar®, catalog number: 3516 )
- Normal human fibroblasts (IMR-90) (ATCC, catalog number: CCL-186 )
- DMEM
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140095 )
- Penicillin-streptomycin (10,000 U/ml, 10,000 µg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
- Amphotericin B (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
- RPMI 1640 medium, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 11835030 )
- DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
- NP-40
- Sodium chloride (NaCl)
- Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
- Phosphatase inhibitor (EMD Millipore, catalog number: 524625 )
- α-SMA antibody (American Research Products, catalog number: 03-61001 )
- β-actin antibody (Sigma-Aldrich, catalog number: A5441 )
- Tween 20
- Fibroblast culture media (see Recipes)
- Lysis buffer (see Recipes)
Equipment
- Cell culture incubator, 37 °C, 5% CO2: 95% air atmosphere (Thermo Fisher Scientific, FormaTM, model: Direct Heat CO2 Incubator )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Larson-Casey, J. L. and Carter, A. B. (2016). Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression. Bio-protocol 6(22): e2009. DOI: 10.21769/BioProtoc.2009.
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分类
免疫学 > 免疫细胞功能 > 巨噬细胞
生物化学 > 蛋白质 > 活性
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