Glucose Uptake Assay Kit (Colorimetric) (ab136955)

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.

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.

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.

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.

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.

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.

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.

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.

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.

We would recommend that you simply scrape the cells in the extraction buffer and take them out in eppendorf tubes. The heat/cool cycle will then take care of denaturing of enzymes.