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| Product Description |
Glucose transport systems are responsible for transporting glucose across cell membranes. Measuring uptake of 2-deoxyglucose (2-DG), a glucose analog, in tissues and cells is widely accepted as a reliable method to estimate the amount of glucose uptake and to investigate the regulation of glucose metabolism and mechanism of insulin resistance. The 2-DG uptake is commonly determined by using non-metabolized 2-DG labeled with tritium or C14. However, routine use of a radiolabelled probe is costly and requires a tedious special handling procedure.This Colorimetric Glucose Uptake Assay Kit provides a sensitive and non-radioactive assay in tissues or cultured cells. In this assay 2-DG is taken up by glucose transporters, and metabolized to 2-DG-6-phosphate (2-DG6P). The non-metabolizable 2-DG6P accumulates in the cells, and is proportional to glucose uptake by cells. The accumulated 2-DG6P is enzymatically oxidized and generates NADPH, which is specifically monitored by a chromogenic NADPH sensor. The signal can be read by a absorption microplate reader by reading the OD ratio at wavelength 570 nm to 610 nm. Component
Sample experimental plan Instruction Important: This protocol can be used as guidelines to culture 3T3-L1 adipocytes for 2-DG uptake. 1. Plate cells in growth medium at 50,000-80,000 cells/well/100 µL/96-well or 12,500-20,000 cells/well/25 µL/384-well black wall/clear bottom cell culture Poly-D lysine plate for 4-6 hours before experiment. 2. Remove the cell plate from the incubator, aspirate the medium from the wells, and deprive the cells with 100 µL/well (96 well-plate) or 25 µL/well (384 well- plate) serum free medium. Incubate the cells at 37oC, 5% CO2 incubator for 6 hours to overnight. 3. Remove the cell plate from the incubator, aspirate the medium from the wells, and gently wash the cells twice with 100 µL/well 1× KRPH buffer. 4. Add 90 µL/well Glucose Uptake Buffer (Component B) and incubate the cells at 37oC, 5% CO2 incubator for 1 hour. 5. Stimulate with or without insulin or compound of test for 20 min. Add 10 µL/well of the 10× insulin solution to a final concentration of 1 µM or 10× compound solution of test. And also add 10 µL insulin vehicle buffer or compound vehicle buffer to the untreated wells as control, and incubate at 37oC, 5% CO2 incubator for 20 mins. 6. For glucose uptake inhibition study, add 10× Phloretin to a final concentration of 200 uM or inhibitors of test, and incubate at 37oC, 5% CO2 for 2-5 mins. Note:10 mL inhibitor vehicle buffer is suggested to be added to both the insulin treated and untreated wells as control. Phloretin treated cells can be used as positive control. 7. Add 10 µL/well 2-DG solution (Component A) to each well, and incubate at 37oC, 5% CO2 incubator for 20-40 mins. For negative controls, leave some wells untreated with insulin, inhibitor and 2-DG. 8. After treatment, remove solution in each well and gently wash cells 3 times, 100 µL/well with KRPH Buffer to remove the extra 2-DG from the solution. Remove KRPH Buffer from the wells. 9. Add 25 µL/well Acidic Lysis Buffer (Component C) to each well and incubate at 37oC for 20 min to lyse the cells. And the 2DG uptake assay mixture could be prepared in the meantime. 10. Add 25 µL/well Neutralization Buffer (Component D) to each well, mix thoroughly, leave at room temperature for 5-10 minutes to neutralize the cell lysate. 11. Add 50 µL of 2DG Uptake Assay working solution to each well of 2-DG6P standard (Not provided) or cell lysate. 12. Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light. 13. Monitor the absorbance ratio increase at 570/610 nm with an absorbance plate reader. EXAMPLE DATA ANALYSIS AND FIGURES
Figure 1. Measurement of 2DG uptake in differentiated 3T3-L1 adipocytes and 3T3-L1 fibroblasts. Assays were performed with Screen Quest™ Colorimetric Glucose Uptake Assay Kit in a black wall/clear bottom cell culture Poly-D lysine plate using a SpectraMax (Molecular Devices) microplate reader. (A: Negative Control, no insulin no 2-DG treatment. B: 2DG uptake in the absence of insulin. C: 2DG uptake in the presence of 1mM insulin. D: 2DG uptake in the presence of 1mM insulin and 200 mM phloretin. E: 2DG uptake in the presence of insulin 1mM and 5mM D-Glucose.) Solution preparation |
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