Glycerol Assay Kit (Cell-Based) (ab133130)

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Overview

  • Product name

    Glycerol Assay Kit (Cell-Based)
    See all Glycerol kits

  • Detection method

    Colorimetric

  • Sample type

    Cell culture supernatant, Adherent cells, Cell culture media

  • Assay type

    Cell-based

  • Species reactivity

    Reacts with: Mammals, Other species

  • Product overview

    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.

  • Notes

    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.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • Platform

    Microplate reader

Properties

Images

  • Glycerol Assay Kit (Cell-Based) (ab133130)
    Glycerol Assay Kit (Cell-Based) (ab133130)

    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.

     

  • Glycerol Assay Kit (Cell-Based) (ab133130)
    Glycerol Assay Kit (Cell-Based) (ab133130)

    Standard curve: mean of duplicates (+/- SD) with background reads subtracted

References (0)

Publishing research using ab133130? Please let us know so that we can cite the reference in this datasheet.

ab133130 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

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Question

I'm thinking of using your glycerol assay kit (ab133130) to measure glycerol uptake from media by cells expressing glycerol channels. Do you know if the kit has been used for this before?

I'm also wondering whether the kit is compatible with detergent-based cell lysates? Is there an enzyme step in the assay that detergent would cause problems with?

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Answer

this assay provides a tool for studying triglyceride fatty acid cycling and its regulation in adipocytes or hepatocytes. As the cell accumulates lipids and forms lipid drops, the triglycerides stored in these lipid drops can be hydrolyzed into free fatty acids and glycerol which are released into the environment. The amount of glycerol released into the medium is proportional to the triglyceride/fatty acid cycling rate.

This is not the assay you will need if they wish to study glycerol uptake.

The assay is a cell based kit and depends on whole unlysed cells. Detergents may be a problem.

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Question

I am inquiring about the Glycerol Assay Kit (Cell-Based) (ab133130). We are carrying out some studies with chloroquine and are curious about your use as a positive control for lipid droplet formation in this assay. I could not find any publications for the studies cited in the protocol. Could you please send the references to me?

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Answer

Here are a few references.

Brown, M. S., Y. K. Ho, and J. L. Goldstein. "The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters." Journal of Biological Chemistry 255.19 (1980): 9344-9352.

Fedorko, Martha E., James G. Hirsch, and Zanvil A. Cohn. "Autophagic vacuoles produced in vitro I. Studies on cultured macrophages exposed to chloroquine." The Journal of cell biology 38.2 (1968): 377-391.

Stein, Olga, Jack Vanderhoek, and Yechezkiel Stein. "Cholesterol ester accumulation in cultured aortic smooth muscle cells: Induction of cholesterol ester retention by chloroquine and low density lipoprotein and its reversion by mixtures of high density apolipoprotein and sphingomyelin." Atherosclerosis26.4 (1977): 465-482.

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Question

Ab65337: en el protocolo de su web indica que es un kit con el que podemos detectar glicerol libre por fluorescencia a partir de sobrenadantes celulares. ¿sería posible hacerlo a partir de extractos celulares? ¿cúal sería el protocolo de lisis?
Ab1333130: en el protocolo de su web indica que es un kit cell-based para detectar glicerol por absorbancia pero también indica en el protocolo que se partiría de sobrenadantes celulares, ¿valdría para extractos celulares?

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Answer



Ab65337: Este kit puede usarse a partir de extractos celulares. Para ello, comenzar con un número aproximado de ˜2X10^6 células, suspender el pellet en 500ul (o aproximadamente 4 volúmenes) de assay buffer en hielo usando un homogeneizador (10-15 pases) hasta que se haya alcanzado una lisis eficiente, lo cual puede comprobarse al microscopio.

Centrifugar la muestra y recoger el sobrenadante para utilizarse en los ensayos posteriores. Se recomienda probar distintas diluciones de muestra para asegurar que las lecturas caen en el rango linear de la curva de estándares.

Ab1333130: En este caso no hemos probado el kit con extractos celulares, no podemos sugerir un protocolo concreto de lisis, pero pueden utilizar un protocolo estándar de extracción, similar al anterior, y optimizarlo para cada caso.

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Question

Customer kindly contacted us regarding the supernatant used in this kit. Are there reagents which should be included or should not be present when growing these cells?

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Answer

Since this assay is specific for glycerol, all containers used to collect your samples should be glycerol free. Also, do not solubilize any compounds in glycerol and add them to your cell culture.

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