Isolation and Culture of the Islets of Langerhans from Mouse Pancreas
从小鼠胰腺分离和培养胰岛的Langerhans细胞
发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1840 浏览次数: 25995
评审: Ivan ZanoniFrancesca MingozziEmilie Battivelli
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
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Abstract
The islets of Langerhans are clusters of endocrine cells located within the pancreas. Insulin-producing beta cells are the major cell type within islets, with glucagon-producing alpha cells and somatostatin-producing delta cells the other major cell types. The beta cells are the target of immune-mediated destruction in type 1 diabetes (Graham et al., 2012). Failure of beta cell function accompanied by loss of beta cell mass is also a feature of type 2 diabetes (Wali et al., 2013). Therefore studying the biology of pancreatic islets is important to understand the pathogenesis of diabetes and to develop new therapies. Here we describe the isolation of mouse islets. This requires gentle enzymatic and mechanical digestion of the exocrine tissue and density gradient separation (Chong et al., 2004; Liu and Shapiro, 1995; Thomas et al., 1998). We then describe how islets can be cultured whole or dispersed into single cells for use in a variety of in vitro and in vivo analyses. Using this protocol reliably results in the isolation of 200-400 islets, depending on the strain of mouse.
Materials and Reagents
- 50 ml polypropylene conical tubes (Corning, Falcon®, catalog number: 352070 )
- 0.2 µm syringe filter (e.g., Sartorius AG, catalog number: 16534-k )
- Syringe (volume dependent on volume of collagenase P prepared)
- Ice
- 2 ml slip tip syringe (will hold total volume of 3 ml) (BD, catalog number: 302204 )
- 30 G x ½ in. needle (BD, catalog number: 305106 )
- 500 micron mesh (Sefar Pty Ltd., catalog number: 06-500/38 )
Note: cut into approximately 5 cm squares and autoclaved (Figure 1) (see Notes for alternative product)
Figure 1. 500 µm islet mesh from Sefar Pty Ltd and 50 ml tube lid shown for size reference
- Collagenase P (Roche Diagnostics, catalog number: 11213865001 )
Note: It requires testing and optimisation prior to use (see Note 1). - RPMI 1640 medium (Life Technologies, Gibco, catalog number: 21870-076 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21870-076”. - Histopaque®-1077 (Sigma-Aldrich, catalog number: H8889 )
- Hanks balanced salt solution (HBSS) (Life Technologies, Gibco, catalog number: 14175-079 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14175-079”. - Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
- HEPES (Sigma-Aldrich, catalog number: H3375-100G )
- HBSS/Ca/HEPES (see Recipes)
Equipment
- Icebox
- Dissecting microscope [e.g., Stereoscopic zoom microscope, (Nikon Instruments Inc., model: SMZ745 )]
- Fibre optic light source (e.g., AmScope Dual Goose-neck Fiber-Optic Illuminator, model: HL150-AY )
- Forceps [e.g., Graefe forceps, 100 mm, curved (ProSciTech Pty Ltd., catalog number: T131C-2 )]
- Scissors [e.g., Iris scissors, 115 mm, straight (ProSciTech Pty Ltd., catalog number: T097-2 )]
- Clamp [e.g., 150 mm spencer wells forceps, straight (ProSciTech Pty Ltd., catalog number: T124 )]
- Bench-top centrifuge at room temperature, no brake required (e.g., Thermo Fisher Scientific, HeraeusTM, model: Multifuge X1R )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Graham, K. L., Fynch, S., Papas, E. G., Tan, C., Kay, T. W. and Thomas, H. E. (2016). Isolation and Culture of the Islets of Langerhans from Mouse Pancreas. Bio-protocol 6(12): e1840. DOI: 10.21769/BioProtoc.1840.
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分类
免疫学 > 免疫细胞分离 > 维持和分化
细胞生物学 > 细胞分离和培养 > 细胞分离
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