UA-Glo® Glucose-Uptake Luminescence Detection Kit

Glucose uptake in mammalian cells can be measured by detecting intracellularly produced 2-deoxyglucose-6-phosphate (2DG6P). Mammalian cell glucose metabolism does not produce 2DG6P. After adding 2-deoxyglucose (2DG) to the cell culture medium, 2DG is transported into the cells by glucose transporters and rapidly phosphorylated intracellularly by hexokinase to generate 2DG6P. Intracellular enzymes cannot further modify 2DG6P, and the membrane-impermeability of 2DG6P causes it to accumulate within the cells. Therefore, detecting intracellular 2DG6P allows for the measurement of cellular glucose uptake.

The UA-Glo® Glucose Uptake Luminescence Assay Kit detects 2DG6P levels through a coupled reaction involving glucose-6-phosphate dehydrogenase, reductase, and luciferase. Glucose-6-phosphate dehydrogenase acts on 2DG6P to reduce NAD to NADH, while reductase utilizes NADH to reduce the reductase substrate (also a luciferin precursor) into luciferin. Finally, luciferin is acted upon by luciferase to produce luminescence, which is detected, with the luminescence intensity being proportional to the 2DG6P content. The kit also includes a stop buffer and a neutralization buffer. The stop buffer contains hydrochloric acid and detergent, which rapidly lyse cells, halt cellular metabolism, inactivate intracellular enzymes, and degrade reduced NAD(P)H. This treatment ensures that intracellular enzymes and reduced NAD(P)H do not interfere with subsequent 2DG6P detection. The neutralization buffer adjusts the pH to optimize conditions for the 2DG6P detection reagents.

The UA-Glo® Glucose Uptake Luminescence Assay Kit is a homogeneous assay reagent with high signal-to-noise ratio, excellent reproducibility, and stability, making it suitable for high-throughput sample processing.

UA-Glo® Glucose Uptake Luminescence Assay Kit components and specifications are as follows. The detectable number of reactions for a 96-well plate is calculated based on adding 50 μL of sample, 25 μL of stop buffer, 25 μL of neutralization buffer, and 100 μL of 2DG6P detection reagent per well.

UA-Glo® The UA-Glo® Glucose Uptake Luminescence Detection Kit detects -6-glucose-6-phosphate dehydrogenase, reductase, and luciferase coupled reaction to measure 2DG6P levels. The luciferase reaction exhibits extremely high sensitivity, and pH, temperature, and experimental system settings significantly affect the reaction. Under most experimental conditions, it is not recommended to directly compare absolute sample readings. It is recommended to include the same reference compound and vehicle control on each assay plate. Experimental sample readings should be normalized against the reference compound and vehicle control readings before data processing and comparison. Alternatively, a 2DG6P standard processed in the same manner as the samples can be included on each assay plate to quantify the 2DG6P content in the samples.

UA-Glo® Glucose Uptake Luminescence Detection Kit linear detection limit for 2DG6P in cell lysates is up to 30μM. If sample dilution is required, users should optimize the dilution factor based on their specific experiments.

The following protocol uses a 96-well plate as an example to demonstrate glucose uptake detection. For other plate formats, refer to the attached table for the recommended volumes of 2DG, Stop Buffer, and Neutralization Buffer.

1)Seed experimental cells at an appropriate density in a 96-well white clear-bottom cell culture plate

2)Perform cell treatments as required by the experiment, followed by glucose uptake detection

3)Glucose in the culture medium will interfere with the detection results. If the medium contains glucose, discard the medium and wash the cells with 100 μL PBS 1-3 times to thoroughly remove residual glucose, then discard the final PBS wash. Slowly removing the medium and PBS can more effectively eliminate glucose. If the medium is glucose-free, you can prepare a high-concentration 2DG solution in Step 4 (e.g., 10mM) and add it directly to the medium to achieve a final concentration of 1mM

4)Prepare a PBS-based 1mM 2DG solution, add 50μL per well to the assay plate from Step 3, and incubate at room temperature (22℃-25℃) for 10min. The incubation time may need to be optimized based on specific experiments

5)Add 25μL Stop Reagent per well, and mix by shaking for 15sec

6)Add 25μL Neutralization Reagent per well, and mix by shaking for 15sec

7)One hour before use, prepare the 2DG6P Detection Reagent according to Steps 8/9/10 . Allowing the prepared 2DG6P Detection Reagent to sit at room temperature for 1hr before use can minimize the detection background

8)Thaw the Luciferase Detection Solution at room temperature, invert to mix, and aliquot the required volume for the experiment, equilibrating it to room temperature. Aliquot the remaining Luciferase Detection Solution as needed and store at -80℃. The Luciferase Detection Solution should not be subjected to more than 3 freeze-thaw cycles

9)Thaw the Reductase Substrate at room temperature and keep on ice. Immediately aliquot any remaining Reductase Substrate after preparing the 2DG6P Detection Reagent as needed, and store at -80℃. The Reductase Substrate should not be subjected to more than 3 freeze-thaw cycles

10)Preparation of 2DG6P Detection Reagent: Add the Reductase Substrate to the Luciferase Detection Solution at a 200x dilution to prepare the 2DG6P Detection Reagent. Prepare the 2DG6P Detection Reagent fresh as needed; storage is not recommended

11)Add 100 μL 2DG6P Detection Reagent per well to the assay plate from Step 6, and mix by shaking for 15sec

12)Incubate at room temperature for 0.5-3hr. The signal will continue to increase until all 2DG6P is consumed

13)Read the luminescence signal using a luminometer or multi-mode plate reader

Attached Table: Recommended volumes of each component for different plate formats:

1) Different batches are not recommended for mixed use

2) Without strict validation, it is not recommended to change the dosage of detection reagents

3) For research use only