製品の概要

  • 製品名

    Glucose Uptake Assay Kit (Fluorometric)
    Glucose キット 製品一覧
  • 検出方法

    Fluorescent
  • サンプルの種類

    Adherent cells, Suspension cells
  • アッセイタイプ

    Quantitative
  • 検出感度

    = 0.05 nmol/well
  • 全工程の試験時間

    2h 00m
  • 種交差性

    交差種: Other species, Mammals
  • 製品の概要

    Glucose Uptake Assay Kit (Fluorometric) ab136956 is a highly sensitive and easy to use non-radioactive kit which can detect glucose uptake as low as 50 pmol/well in a variety of cell types.


    2-deoxyglucose (2-DG) is used in glucose uptake assay protocols because of its structural similarity to glucose. 2-DG is taken up by glucose transporters and metabolized to 2-DG-6-phosphate (2-DG6P). 2-DG6P cannot be further metabolized, and thus accumulates within cells. The accumulated 2-DG6P is directly proportional to 2-DG (or glucose) uptake by cells. In this assay, the accumulated 2-DG6P is enzymatically oxidized and coupled to a probe, which generates fluorescence in the presence of NADPH.


    Glucose uptake assay protocol summary:
    - prepare cells with suitable glucose starvation / uptake stimulation depending on experimental set-up
    - add 2-DG to cells and incubate for 20 mins at 37ºC
    - wash cells with PBS to remove exogenous 2-DG
    - lyse cells with extraction buffer and repeated pipetting
    - freeze/thaw lysates and optionally heat at 85ºC for 40 min
    - cool on ice for 5 min
    - add neutralizing buffer, spin and retain supernatant
    - add supernatants and standards to wells
    - add reaction mix and incubate for 40 min at 37ºC

  • 特記事項

    If you want a more sensitive assay, we recommend using Glucose Uptake Assay Kit (Colorimetric) (ab136955), which contains an amplification step that allows the kit to detect < 10 pmol/well.

  • 試験プラットフォーム

    Microplate reader

製品の特性

  • 保存方法

    Store at -20°C. Please refer to protocols.
  • 内容 ラベル 100 tests
    2-Deoxyglucose Purple 1 x 1ml
    2-DG Uptake Assay Buffer WM 1 x 10ml
    2-DG6P Standard (Lyophilized) Yellow 1 vial
    Enzyme Mix (lyophilized) Green 1 vial
    Extraction Buffer NM 1 x 17ml
    Neutralizing Buffer Clear 1 x 1ml
    PicoProbe Blue 1 x 0.2ml
  • 研究分野

  • 関連性

    Glucose (C6H12O6; FW: 180.16) is a ubiquitous energy source in most organisms, from bacteria to humans. The breakdown of carbohydrates produces mono- and disaccharides, most of which is glucose. Through glycolysis and TCA (citric acid cycle), glucose is oxidized to eventually form CO2 and water, generating the universal energy molecule ATP. Glucose is a primary source of energy for the brain and a critical component in the production of proteins and in lipid metabolism and therefore measurement of glucose level is a key diagnostic parameter for many metabolic disorders.

画像

  • Glucose Uptake measured in 3T3-L1 Adipocytes; I = Insulin.
  • Standard curve: mean of duplicates (+/- SD) with background reads subtracted
  • 2-DG6P Standard curve (a) and 2-DG uptake in Human adipocytes (b) , HeLa Cells (c) and 3T3-L1 cells (d) respectively. I=Insulin; P=Phloretin.
  • Accumulated 2-DG6P is enzymatically oxidized and coupled to the picoprobe, which generates fluorescence in the presence of NADPH.

プロトコール

参考文献

This product has been referenced in:

  • Mustroph J  et al. Empagliflozin enhances human and murine cardiomyocyte glucose uptake by increased expression of GLUT1. Diabetologia 62:726-729 (2019). Read more (PubMed: 30694352) »
  • Valzania L  et al. Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti. Proc Natl Acad Sci U S A 115:457-465 (2018). Read more (PubMed: 29298915) »
See all 5 Publications for this product

レビューと Q&A

1-3 of 3 Abreviews or Q&A

Question
Answer


The lab let me know that they have optimized the glucose uptake assay with the KRPH buffer and would not be able to comment on how other buffers might work with it as this has not been tested.

Alternative buffers such as plain HEPES buffer would not be sufficient.

Thus, it is recommended to use KRPH buffer.

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Answer

Thank you for making us aware of this issue. The enzyme mix was missing from the recipe for reaction mix A on page 9. This has been fixed in the current protocol. Please use the protocol included with the kit or the one available on the webpage for this product: https://www.abcam.com/index.html?datasheet=136956

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Answer

Merci de nous avoir contactés.

Il y a en effet une erreur sur le protocole. Voici ce qu'il faut comprendre :

"Treat cells with desired method. For 3T3-L1 adipocytes, for example, seed cells at a density of ˜1500 cells per well in a 96 well plate and differentiate to mature adipocytes, maintain for another 4 days prior to use. To assay glucose uptake, wash adipocytes twice with PBS and starve overnight in 100 µl serum free adipocyte medium (to increase glucose uptake). Wash cells 3X with PBS. Starve the cells for glucose by preincubating with 100 µl Krebs-Ringer-Phosphate-Hepes (KRPH) buffer containing 2 % BSA for 40 min. Stimulate with or without insulin (1 μM) for 20 min to activate glucose transporter. Add 10 µl of 10 mM 2-DG, mix and incubate for 20 min."

Désolé pour ces erreurs qui seront corrigées dans les plus brefs délais.

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

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