Glucose Uptake Assay Kit (Colorimetric) (CAT#: CB-P139-K) Datasheet

Product Type
Kit
Description
The glucose uptake assay kit (colorimetric) is a highly sensitive and easy-to-use non-radioactive assay kit that can detect glucose uptake as low as 10 pmol/well in various cell types.


Since 2-deoxyglucose (2-DG) is similar in structure to glucose, it is used in the glucose uptake measurement protocol. 2-DG is absorbed by glucose transporter and metabolized to 2-DG-6-phosphate (2-DG6P). 2-DG6P cannot be further metabolized, so it will accumulate in the cell. The accumulated 2-DG6P is directly proportional to the 2-DG (or glucose) uptake by the cells. In this test, 2-DG6P is oxidized to produce NADPH, the level of which can be determined by the enzyme cycle amplification reaction.
Applications
Functional Studies
Size
1 kit
Detection Method
Colorimetric
Sensitivity
<= 0.01 nmol/well
Sample
Adherent cells, Suspension cells
Species Reactivity
Mammals
Protocol Length
3h 00m
Components
2-Deoxyglucose 1 x 1ml
2-DG6P Standard (Lyophilized) 1 vial
Assay Buffer 1 x 25ml
Enzyme Mix (Lyophilized) 1 vial
Extraction Buffer 1 x 17ml
Glutathione Reductase (Lyophilized) 2 vials
Neutralizing Buffer 1 x 2.5ml
Recycling Mix(Lyophilized) 1 vial
Substrate 2 vials
Requires
Microplate reader
Handling Advice
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 37°C
- wash cells with PBS to remove exogenous 2-DG
- lyse cells with extraction buffer and repeated pipetting
- freeze/thaw lysates and heat at 85°C 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 37°C
- add extraction buffer and heat to 90°C 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 37°C
Storage
Please refer to protocols.
Shelf Life
12 months
Research Use
For research use only. Not for human or drug use

Figure 1 Standard curve and example data.
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.


Figure 2 Functional Studies
Glucose uptake in 3T3-L1 adipocytes stimulated with insulin (I).


Figure 3 Assay Procedure
Step A: 2-DG oxidation to generate NADPH; Step B: NADPH recycling amplification Reaction.

All products and services are for Research Use Only. Do Not use in humans.

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