Glucose Uptake Assay Kit (Fluorometric) (ab136956)

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Overview

  • Product name

    Glucose Uptake Assay Kit (Fluorometric)
    See all Glucose kits

  • Detection method

    Fluorescent

  • Sample type

    Adherent cells, Suspension cells

  • Assay type

    Quantitative

  • Sensitivity

    = 0.05 nmol/well

  • Assay time

    2h 00m

  • Species reactivity

    Reacts with: Other species, Mammals

  • Product overview

    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

  • Notes

    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.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 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

  • Research areas

  • Relevance

    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.

Associated products

Images

  • Functional Studies - Glucose Uptake Assay Kit (Fluorometric) (ab136956)
    Functional Studies - Glucose Uptake Assay Kit (Fluorometric) (ab136956)
    Glucose Uptake measured in 3T3-L1 Adipocytes; I = Insulin.
  • Functional Studies - Glucose Uptake Assay Kit (Fluorometric) (ab136956)
    Functional Studies - Glucose Uptake Assay Kit (Fluorometric) (ab136956)
    Standard curve: mean of duplicates (+/- SD) with background reads subtracted
  • Example data
    Example data
    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.
  • Assay Procedure
    Assay Procedure

    Accumulated 2-DG6P is enzymatically oxidized and coupled to the picoprobe, which generates fluorescence in the presence of NADPH.

Protocols

References

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

Publishing research using ab136956? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-3 of 3 Abreviews or Q&A

Question

why should KRPH be used?
can e.g. HBSS or PBS or other buffers (without glucose) be used?

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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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Question

Customer contacted us as the protocol which they found on Google was not correct. Reaction mix A only amounts to 48ul when it says it should be 50ul.

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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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Question

Madame, Monsieur,
J'ai commandé le kit de glucose uptake chez vous (ref 136956). Or j'ai des
questions vis à vis du protocole. Dans le paragraphe de sample preparation, vous parlez de 100GI serum free adipocyte medium puis de 100GI KRPH buffer et enfin de 10GI 2-DG. J'aimerai savoir à quoi correspond 100GI, une mesure comme des µL, un tampon?
d'autre part à aucun moment dans le protocole vous stipulez à quel moment mettre enzyme mix et en quelle quantité.
Pourriez-vous répondre à mes questions afin que je puisse réaliser mon expérience dans les meilleures conditions.
Cordialement

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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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