Isolation and Primary Culture of Adult Mouse Cardiac Fibroblasts
成年小鼠心脏成纤维细胞的分离和原代培养
发布: 2016年07月05日第6卷第13期 DOI: 10.21769/BioProtoc.1860 浏览次数: 19010
评审: Jingli CaoAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2014
Abstract
Fibroblasts are often used as a feeder layer for progenitor or stem cells in co-culture systems. In the heart fibroblasts are important for cardiac development, homeostasis, and remodelling. They provide cardiomyocytes and progenitor cells not only with nutrition but also secrete extracellular matrix that forms the microenvironment that ensures cell survival and function. Although different kinds of mouse fibroblasts have been used in co-cultures (embryonic, skin and cardiac fibroblasts) adult mouse cardiac fibroblasts (AMCFs) create the closest microenvironment to the adult murine heart for culturing adult mouse cardiac progenitor cells. This protocol describes the isolation of cardiac fibroblasts from adult mouse hearts as well as their maintenance in culture.
Materials and Reagents
- Falcon tubes (50 ml) (Sarsdtedt)
- Sterile pipets (Sarsdtedt)
- 100 mm cell culture dishes (Sarsdtedt)
- Aluminum foil
- Adult mouse hearts from freshly euthanized mice (C57Bl6/J)
- NaCl (Carl Roth, catalog number: 3957.2 )
- KCl (Carl Roth, catalog number: HNO2.3 )
- Na2HPO4 (Carl Roth, catalog number: 4984.1 )
- KH2PO4 (AppliChem, catalog number: A2946 )
- CaCl2 (AppliChem, catalog number: A3652 )
- MgSO4.7H2O (AppliChem, catalog number: A1037 )
- NaHCO3 (AppliChem, catalog number: A1940 )
- Collagenase II (Worthington, catalog number: LS004177 )
- 2.5% Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 15090-046 )
- DMEM/F12 (Thermo Fisher Scientific, GibcoTM, catalog number: 31331-028 )
- PBS (without CaCl2, MgCl2) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-94 )
- FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
- PenStrep (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
- L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-024 )
- Ascorbic acid (AppliChem, catalog number: A1052 )
- PBS (see Recipes)
- Hanks balanced salt solution (HBSS) (see Recipes)
- Collagenase II stock (see Recipes)
- Digestion buffer (see Recipes)
- Fibroblast medium (see Recipes)
Equipment
- Scalpel, forceps and scissors (Fine Science Tools)
- Autoclaved glass beaker (100 ml) with magnetic stirrer
- Sterile cell culture hood
- Cell culture incubator (Labotect Labor-Technik Göttingen)
- Magnetic stirrer with thermostat (IKA®-Werke GmbH & CO. KG)
- Cell culture centrifuge (Eppendorf, model: 5417R )
- Autoclave
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zafeiriou, M. P., Noack, C. and Zelarayan, L. C. (2016). Isolation and Primary Culture of Adult Mouse Cardiac Fibroblasts. Bio-protocol 6(13): e1860. DOI: 10.21769/BioProtoc.1860.
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分类
细胞生物学 > 细胞分离和培养 > 细胞分离
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