Glucose Production Assay in Primary Mouse Hepatocytes
原代小鼠肝细胞中的葡萄糖生成分析
发布: 2012年11月05日第2卷第21期 DOI: 10.21769/BioProtoc.284 浏览次数: 26481
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参见作者原研究论文
The authors used this protocol in:
Mar 2012
Abstract
Hepatic glucose production is a primary determinant of fasting hyperglycemia in type 2 diabetic patients. Glucagon-cAMP-PKA pathway increases, but insulin-PI3 kinase-Akt pathway suppresses glucose production. This assay aims to evaluate the ability of isolated mouse hepatocytes to release newly synthesized glucose mainly from lactate and pyruvate as the substrates (i.e. gluconeogenesis) under basal, cAMP-, or cAMP plus insulin-treated condition.
Materials and Reagents
- Primary mouse hepatocytes
- Medium199 (Life Technologies, Invitrogen™, catalog number: 11150-059 )
- Fetal bovine serum (FBS)
- bicinchoninic acid (BCA)
- Penicillin-Streptomycin, Liquid (Life Technologies, Invitrogen™, catalog number: 15140-122 )
- PBS+/+ (Sigma-Aldrich, catalog number: D8662 )
- Dulbecco’s modified eagle’s medium (DMEM), without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate, powder (Sigma-Aldrich, catalog number: D5030 )
- Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
- Sodium L-lactate (Sigma-Aldrich, catalog number: L7022 )
- Sodium pyruvate (Life Technologies, Invitrogen™, catalog number: 11360-070 )
- 100 mM MEM sodium pyruvate solution (100x), liquid (Life Technologies, Invitrogen™, catalog number: 11360-070 )
- 200 mM L-Glutamine (100x), liquid (Life Technologies, Invitrogen™, catalog number: 25030-081 )
- 1 M HEPES buffer solution (Life Technologies, Invitrogen™, catalog number: 15630-080 )
- pCPT-cAMP (Sigma-Aldrich, catalog number: C3912 )
- Insulin (Sigma-Aldrich, catalog number: I9278 )
- Autokit glucose (Wako, catalog number: 439-90901 )
- BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23227 )
- HEPES
- Glucose production buffer (see Recipes)
Equipment
- iMark Microplate Absorbance Reader (Bio-Rad, catalog number: 168-1135 )
- BD BioCoat™Collagen I 6-well Plates (BD Biosciences, catalog number: 356400 )
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Matsumoto, M. and Sakai, M. (2012). Glucose Production Assay in Primary Mouse Hepatocytes. Bio-protocol 2(21): e284. DOI: 10.21769/BioProtoc.284.
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分类
生物化学 > 糖类 > 葡萄糖
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