I would like to measure glucose concentration in plasma/media before and after glucose uptake process in cells.
Glucose concentration is easily measured by a Hexokinase assay linked to the reduction of NAD+. Whereby Glucose+ATP is converted to glucose-6-phosphate and ADP, catalysed by hexokinase. In the presence of NAD+, glucose-6-phosphate is converted to 6-Phosphogluconate and NADH, catalysed by glucose-6-phosphate dehydrogenase. The presence of NADH can be measured spectrophotometrically at 340 nm and is proportional to the original glucose concentration. Glucose in your unknown samples can be quantified from a standard curve derived from known glucose concentrations.
See Glucose (HK) Assay Kit from Sigma (product code GAHK-20).
Bets wishes and good luck
Jonathan
Anat,
Jonathan has already given you a good run down on the enzyme linked method using NADH absorption at 340nm. The only potential issue I see is the sensitivity of the method, which may limit your ability to measure small differences glucose concentrations. 1mM NADH at 340nm would yield you an absorbance equivalent of 6.2 (1cm path length).
If your cells consume sufficient glucose to cause a change in glucose concentration in the range of 20µM-200µM during your observation period, you should be able to get an accurate measurement. However, if the observation period is short or cells are relatively few for a given volume of medium, you may run into difficulty in discerning the difference reliably. If that is the case, use fluorescence based method that is accurate in lower range of concentration (3µM-50µM). Invitrogen used to sell a kit, I presume they still do. The method involves activating fluorescent resorufin, which has absorption maximum at 571 nm and emission maximum at 585 nm.
Best,
Hello
Agree with researchers , and Suggested Use :
# glucose-assay-kit-ab65333 in abcam
http://www.abcam.com/glucose-assay-kit-ab65333.html
# glucose-detection-kit-ii-ab102517
http://www.abcam.com/glucose-detection-kit-ii-ab102517.html
Good Luck
Tausif,
Is this the kit?
https://www.thermofisher.com/order/catalog/product/A22189
Did you use it? was it reliable?
Anat,
I believe that's the one!
The method is reliable as long as you follow due precautions typical with any fluorescence based detection with appropriate controls. Rather than using endpoint measurements, if your spectrofluorometer permits kinetic measurements, you could improve the quality of data by using the initial linear rate of reaction.
Best,
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