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Glycerol Assay Kit (Cell-Based) (ab133130) provides a convenient tool for studying triglyceride/fatty acid cycling and its regulation in adipocytes or hepatocytes. This kit will allow investigators to screen compounds involved in lipid storage and metabolism. Chloroquine is included in the kit as a positive control for screening compounds that induce lipid droplet accumulation and free glycerol release from hepatocytes.
In mammals, triglycerides are constantly synthesized from fatty acids and segregated into cytosolic lipid droplets, mainly in adipocytes, as the major energy storage depot. During fasting, triglycerides stored in adipose tissue and liver are hydrolyzed by hormone-sensitive lipase and adipose triglyceride lipase to produce free fatty acids and glycerol. Triglyceride/fatty acid cycling is important in metabolic regulation and heat production, and is highly regulated by enzymes such as phosphenolpyruvate carboxykinase (PEPCK) and lipases. Quantitative changes in the triglyceride/fatty acid cycle have been related to the increased metabolic rate of cachectic patients with esophageal cancer and to metabolic syndrome. Abnormal triglyceride accumulation in the form of lipid droplets can occur in adipocytes and/or hepatocytes of obese mammals. In vitro, dramatic lipid accumulation can be observed in well-differentiated 3T3-L1 cells, or HepG2 cells treated with steatosis-inducing compounds such as chloroquine. Triglycerides stored in these lipid droplets can be hydrolyzed into free fatty acids and glycerol which are subsequently released into the surrounding environment. The amount of glycerol released into the medium is proportional to the triglyceride/fatty acid cycling rate.
|Chloroquine Positive Control (25 mM)||1 vial|
|Free Glycerol Assay Reagent (10X)||2 vials|
|Glycerol Standard Solution||1 vial|
Glycerol release from HepG2 cells treated with chloroquine.
HepG2 cells were seeded at a density of 104 cells/well in a 96-well plate and grown overnight in a 37°C incubator. The next day, cells were treated with vehicle or different doses of chloroquine for 24 hours. At the end of this incubation, supernatants were collected and analyzed for free glycerol according to the procedure described in the protocol.
Standard curve: mean of duplicates (+/- SD) with background reads subtracted
ab133130 has not yet been referenced specifically in any publications.
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