AMPK Methods and Protocols

2 Materials and Reagents Prepare all solutions using ultrapure water (mQ water; sensitivity 18 MΩ-cm at 25C) and analytical gr ...

2.2 Culture Medium and Extraction Buffer Culture medium for primary hepatocytes: M199 medium con- taining 10% (v/v) FCS, 0.1% ( ...

container. Add 1.2 g of tris(hydroxymethyl)aminomethane and 190 mg of EGTA. Mix and adjust pH to 7.2 with HCl. Make up to 500 ml ...

CCCP: 100 mM carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Dissolve 20.5 mg of CCCP in 1 ml of DMSO. Aliquot (100μl) in 0.5 ...

Start the acquisition of the basal oxygen consumption rate (JO 2 ) until reaching a steady state (usually ~1–2 min, Fig.2). Add ...

3.1.2 Seahorse The regulation of mitochondrial oxidative phosphorylation (OXPHOS) can be assessed in intact adherent cells (e.g. ...

Port B (CCCP): prepare 2.4 ml of intermediate solution by adding 36μl of CCCP 1:100 (stock: 100 mM; 1:100 stock: 1 mM; final co ...

Add 20μl of ADP (stock: 100 mM; final concentration: 1 mM) and measure theJO 2 coupled to oxidative phosphorylation (state 3) d ...

Wash carefully the oxygraph chamber after each measure for three times with mQ water, then three times with 70% ethanol and aga ...

Wash carefully the oxygraph chamber after each measure for three times with mQ water, then three times with 70% ethanol and aga ...

MCDB-131 medium or KB cells (10^7 cells/ml) in RPMI 1640 medium, as previously reported [16, 17]). After ~30 s of equilibration, ...

Acknowledgments Guigas’ group is supported by funding from the European Federa- tion for the Study of Diabetes (EFSD/Lilly Resea ...

mitochondrial respiratory chain complex Cell Transplant 16(4):409–419 Taleux N, Guigas B, Dubouchaud H, Moreno M, Weitzel JM, G ...

Chapter 19 Study of AMPK-Regulated Metabolic Fluxes in Neurons Using the Seahorse XFe Analyzer Claudia Marinangeli, Je ́rome Klu ...

upregulating the glycolytic flux via 6-phosphofructo-2-kinase phosphorylation [4–7]. Additionally, AMPK is responsible for sti- ...

2.1 Equipment 1. Seahorse XFe24 Analyzer. Seahorse XFe24 cell culture plates and corresponding sensor cartridges (Fig.1a and b) ...

the sensor cartridge (Fig.1b and c). For glycolysis assay, glucose, oligomycin, and 2-deoxy-D-glucose (2DG) will be sequentially ...

Glucose intermediate solution: 72.5 mM glucose. Dilute 20% glucose stock solution accordingly. The final concentration during t ...

3 Methods This present protocol has been optimized for studying metabolic fluxes in primary neuronal cultures, using a Seahorse ...

Primary Neuronal Culture Preparation Dissect and isolate mouse cortical and/or hippocampal neu- rons using an enzymatic dissoci ...