Cell Meter™ 2-NBDG Glucose Uptake Assay Kit

Protocol summary

1. Prepare cells with your test compounds
2. Add 2-NBDG staining solution
3. Incubate cells  at 37^oC for 20 minutes
4. Remove 2-NBDG staining solution
5. Wash cells with Assay Buffer I
6. Analyze cells using fluorescence microscope or flow cytometer with 530/30 nm filter (FITC channel)

Important notes
Thaw all the components at room temperature before starting the experiment.

Add 5 µL of 2-NBDG (10 mg/mL) (Component A) to 1.5 mL of Assay Buffer I (Component B) and mix well to make 2-NBDG staining solution. Protect from light. Note: This 2-NBDG staining solution is stable for 1 hour at room temperature. As the optimal staining conditions may vary depending on different cell types, it’s recommended to determine the optimal concentration of Component A for each specific experiment.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Add test compounds into the cells and incubate for a desired period of time (such as 24, 48 or 96 hours) in a 37°C, 5% CO₂ incubator. For blank wells (medium without the cells), add the same amount of compound buffer. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density and incubation time. We incubated CHO-K1 cells with 20 mM Glucose for glucose competition assay, and 100 µM Phloretin for GLUTs inhibition assay. See Data Analysis for details.
   
   
2. At the end of the treatment, centrifuge the plate for 5 minutes at 800 rpm with brake off prior to your experiment.
   
   
3. Aspirate the supernatant without disturbing cells.
   
   
4. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of 2-NBDG staining solution. Note: Optimal incubation time will need to be determined for each cell line and for each specific experiment. We incubated CHO-K1 cells at 37^oC with 100 µM 2-NBDG (~34 µg/mL) for 20 minutes to show sufficient glucose uptake. See Data Analysis for details.
   
   
5. At the end of the incubation, centrifuge the plate for 5 minutes at 800 rpm.
   
   
6. Remove 2-NBDG staining solution without disturbing cells.
   
   
7. For fluorescence microscope: Wash cells with Assay Buffer I (Component B) once. Keep cells in 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Assay Buffer II (Component C). Monitor the fluorescence signal using a fluorescence microscope with FITC filter.
   
   
8. For flow cytometer: Detach cells if required using EDTA and resuspend cells in 100 µL/sample of Assay Buffer I (Component B). Monitor the fluorescence signal using a flow cytometer with 530/30 nm filter (FITC channel).