Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes
肝细胞糖酵解和脂肪生成
发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1993 浏览次数: 12027
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参见作者原研究论文
The authors used this protocol in:
Jan 2016
Abstract
Metabolic flux analyses are needed to provide insights into metabolic regulation that occurs in cells. The current protocol describes fast and reproducible methods for determining glycolysis and de novo lipogenesis of hepatocytes. Primary culture of hepatocytes is an ‘in vitro’ model useful to study liver glucose and lipid metabolism (Denechaud et al., 2016). The protocol is divided in 2 parts. Part I: Glycolysis experiment is assessed using the Seahorse extracellular flux (XF) analyser. Glycolysis is determined via the measurement of the extracellular acidification rate (ECAR) of the media, which come predominately from the cellular excretion of lactic acid after the conversion of glucose in pyruvate. Part II: De novo lipogenesis experiment determines the radioactive C14 incorporation in triglycerides (TG) from acetate C14 precursor. After 2 h acetate supplementation to the media lipids are extracted and separated by TLC (Thin Layer Chromatography) prior quantification of newly synthetized TG labelled.
Background
There are different approaches for evaluating glucose and lipid metabolism: metabolite quantification, enzyme activity, and metabolomics... Our protocols focus on metabolic flux analyses of live cells and do not need a metabolomic facility. The Seahorse extracellular flux (XF) analyzer, which is now present in a lot of institution, is a powerful tool for measuring indirectly glycolysis in live cells by determining media pH. Lipogenesis protocol does not need a big investment and is highly reproducible. It could also be determined using tritiated water, which is incorporated by the Fatty Acid Synthase in de novo synthetized lipids.
Materials and Reagents
- 60 mm tissue culture dishes (TPP, catalog number: 93060 )
- XF assay 24 well cartridge and cell culture microplate V7 (Agilent Technologies, Seahorse Bioscience, catalog number: 100850-001 )
- 8-15 weeks old C57BL/6 mouse (Janvier lab)
- Collagen type I, rat tail (EMD Millipore, catalog number: 08-115 )
- M199 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 41150020 )
- Trypsin (2.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
- Fetal bovine serum (FBS)
- DMEM without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate, powder, suitable for cell culture (Sigma-Aldrich, catalog number: D5030-10X1L )
- D-(+)-glucose solution (Sigma-Aldrich, catalog number: G8769 )
- L-glutamine, 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
- XF calibrant solution (Agilent Technologies, Seahorse Bioscience, catalog number: 100840-000 )
- PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
- Sodium chloride (NaCl) (AppliChem, catalog number: A3597 )
- Phenol red sodium salt (Sigma-Aldrich, catalog number: P5530 )
- Tris, pH 7.5 (Applichem, catalog number: A1379 )
- EDTA (Sigma-Aldrich, catalog number: E6758 )
- Triton X-100 (Sigma-Aldrich, catalog number: X100 )
- Protease inhibitor (Sigma-Aldrich, catalog number: P8340 )
- Glycolysis media (see Recipes)
- 9x glucose solution (see Recipes)
- Lysis Buffer (see Recipes)
Equipment
- CO2-free incubator set to 37 °C (Memmert or other suppliers)
- 5% CO2 incubator set to 37 °C (SalvisLab or other suppliers)
- Centrifuge
- XF 24 extracellular flux analyser (Seahorse Bioscience)
- Cell culture hood (Vitaris or other suppliers)
- pH meter (Mettler Toledo or other suppliers)
- Cell counter (Thermo Fisher Scientific or other suppliers)
Software
- Seahorse software (XFReader 24 version 1.6 supply with the XF 24 extracellular flux analyser)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Denechaud, P. and Fajas, L. (2016). Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes. Bio-protocol 6(21): e1993. DOI: 10.21769/BioProtoc.1993.
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分类
细胞生物学 > 细胞新陈代谢 > 脂质
细胞生物学 > 细胞新陈代谢 > 糖类
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