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

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