製品の概要

  • 製品名
    Glucose Uptake Assay Kit (Colorimetric)
    Glucose uptake キット 製品一覧
  • 検出方法
    Colorimetric
  • サンプルの種類
    Adherent cells, Suspension cells
  • アッセイタイプ
    Cell-based (quantitative)
  • 検出感度
    <= 0.01 nmol/well
  • 全工程の試験時間
    3h 00m
  • 種交差性
    交差種: Other species, Mammals
  • 製品の概要

    Glucose Uptake Assay Kit (Colorimetric) (ab136955) is a highly sensitive and easy to use non-radioactive assay kit which can detect glucose uptake as low as 10 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 2-DG6P is oxidized to generate NADPH, the level of which can be determined by an enzymatic recycling amplification reaction.


    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 heat at 85ºC for 40 min
    - cool on ice for 5 min
    - add neutralizing buffer, spin and transfer supernatant to new tubes
    - add supernatants and standards to wells
    - add reaction mix A and incubate for 1 hr at 37ºC
    - add extraction buffer and heat to 90ºC for 40 min
    - cool on ice for 5 min and add neutralizing buffer
    - add reaction mix B
    - analyze every 2-3 mins on microplate reader in kinetic mode at 37ºC

  • 特記事項

    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • 試験プラットフォーム
    Microplate reader

製品の特性

画像

  • Glucose uptake in 3T3-L1 adipocytes stimulated with insulin (I). 3T3-L1 adipocytes were differentiated using:

     

    Dexamethasone ab120743 (1mM, 1:1000)

    IBMX ab120840 (11.5 mg/mL, 1:100)

    Insulin ab123768 (1 mg/mL, 1:1000)

  • 2-DG6P Standard curve (a) and 2-DG uptake in 3T3-L1 cells (b), Human adipocytes (c) and HeLa cells (d) respectively. I=Insulin; P=Phloretin.
  • Step A: 2-DG oxidation to generate NADPH; Step B: NADPH recycling amplification Reaction.

プロトコール

参考文献

This product has been referenced in:
  • Li H  et al. Fibroblast growth factor 21 increases insulin sensitivity through specific expansion of subcutaneous fat. Nat Commun 9:272 (2018). Read more (PubMed: 29348470) »
  • Wu D  et al. HIF-1a is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium. Elife 6:N/A (2017). Read more (PubMed: 28556776) »
See all 12 Publications for this product

レビューと Q&A

1-10 of 12 Abreviews or Q&A

Answer

We would not recommend storing samples or stopping and storing samples during the assay procedure. However, if absolutely necessary the samples can be stored as cells at -80C before the addition of the extraction buffer to avoid any complications due to storage.

This kit is a very sensitive and multi-step assay using several enzymes. It is not recommended to stop and store cells in the middle of the assay since the enzymes might not work well after freezing and thawing. Once the cells are lysed using the extraction buffer, the entire assay must be completed to ensure best results.

Read More

Answer

We have only tested this kit with human sample. However, we predict that this kit can work with a wide range of mammals including mouse.

Read More

Question
Answer

Even though this kit has not been tested on bacterial samples, in principle it should work with samples from a wide range of mammals and bacteria. The method of detection is based on a biochemical reaction rather than an immunological reaction.

Read More

Answer

Thank you for your enquiry.

I can confirm that the 2DG in the kit can be dissolved in water and is soluble up to 100 mM in water.

I hope this will be helpful. If you have any further questions, please do not hesitate to contact me.

Read More

Answer

Serum starvation is required because serum can provide carbon source from which the cells can make glucose.

There should be no insulin in the media during starvation. After starvation, cells are stimulated with insulin to uptake glucose via the GLUT transporters.

Read More

Glucose uptake assay of C. elegans

Excellent Excellent 5/5 (Ease of Use)
Abreviews
This kit worked well for C. elegans assay. We tried several 1) worm amount, 2) 2DG concentrations and uptake time, 3) sample dilutions. Here we provide the optimal protocol that we found in our trial.

1. Collect 50 ul of young adult worms for each sample in 1.5 mL tube (about 1/2 worms on 9 cm dish)
2. Resuspend worms in M9 buffer (or other saline for worm) and incubate for 1 hrs
3. Remove buffer and incubate worms in 0.5 mM 2DG in M9 buffer for 2 hr
4. Wash worms with ice-cold PBST for 3 times
5. Add 80 ul of Extraction buffer, pipette up and down, and freeze
6. Thaw samples at 85 C for 40 min
7. Add 10 ul Neutralization buffer and spin briefly
8. Dilute supernatant 1/8 times with Assay buffer
9. Add 10 ul Reaction mix A, incubate at 37 C for 1 hr
10. Add 90 ul Extraction buffer and heat at 85 C for 40 min
11. Add 12 ul Neutralization buffer
12. Transfer standards and samples to a 96 well plate
13. Add 38 ul Reaction mix B, pipette up and down
14. Measure absorbance at OD412 nm at 37 C every 5 min until 100pmol/well standard reaches OD412 nm = 1.5 - 2.0

Shun Kitaoka

Verified customer

投稿 Mar 12 2014

Answer

To do the differentiation in the flask, the washing steps after differentiation will have to be done in tubes which can be spun down to collect cells. Then the starvation step has to be done again in a flask and the following washes in a tube, spinning down the tube after each wash to collect cells. Subsequent steps of stimulation with insulin, 2DG incubation have to be done in the tubes. Finally, cell aliquots can be added to each well, then assay reagents can be added and measured. Optimization might be necessary to carry out all the steps in flasks/tubes without loss of material and to get the best, most accurate measurements.

Read More

Question
Answer

The kit is based on the fact that DTNB is converted with enzyme to TNB which has absorption maxima at 412. As all the dyes have bell shaped curves so wavelengths higher or lower than maxima should also be suitable; individual absorption maxima curves can be checked for selections of instruments.

Read More

Answer



You need to increase the volumes of all reagents used such that the cells are covered and are not exposed to the air to dry out. For example, for the serum starvation, instead of using the 100 µl media for covering cells in the 96 well plate, I would use about 2 ml media for the 3.5 cm plate.

Read More

Question
Answer

I am happy to confirm that this kit can be used multiple times.

I recommend to store all the kit components at the recommended temps and in aliquots when possible.

Please take note that repeated freeze/thaw cycles will damage the components.

Read More

1-10 of 12 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

登録