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AAT Bioquest

Screen Quest™ Fluorimetric Glucose Uptake Assay Kit

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. AAT Bioquest's Screen Quest™ Fluorimetric Glucose Uptake Assay Kit provides a sensitive and non-radioactive assay in 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 fluorogenic NADPH sensor. The signal can be read by a fluorescence microplate reader.

Example protocol

AT A GLANCE

Protocol summary

  1. Plate cells and treat the cells as desired
  2. Add 10 µL/well 2-DG and incubate at 37oC for 20-40 minutes
  3. Wash cells and lyse cells
  4. Add 50 µL/well of 2-DG Uptake Assay working solution
  5. Incubate at RT for 30 to 120 minutes
  6. Monitor fluorescence increase at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment. The above mentioned protocol is for one 96-well plate.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

KRPH Buffer stock solution (1X):
Add 20 mL of KRPH Buffer (5X) (Component H) to 80 mL of deionized water and mix them well. Note: 50 mL volume of 1× KRPH Buffer is enough for approximately one 96-well plate. Prepare the needed volume proportionally. Store the unused 1×KRPH at 4oC or -20oC.

PREPARATION OF WORKING SOLUTION

1. NADP working solution:
Add 100 µL of H2O into the vial of NADP (Component G) and mix them well.

2. Enzyme Probe working solution:
Add 5 mL of Assay Buffer (Component F) into the bottle of Enzyme Probe (Component E) and mix them well.

3. 2-DG Uptake Assay working solution:
Add 100 µL of NADP working solution into the Enzyme Probe working solution and mix them well. Note: These quantities are good for one 96-well plate.

SAMPLE EXPERIMENTAL PROTOCOL

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 mins. 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 min. 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 min. 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 mins 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 2DG6P 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 fluorescence increase with a fluorescence plate reader at Ex/Em = 530-570/590-600 nm (optimal Ex/Em = 540/590 nm, cut off at 570 nm).

Citations

View all 11 citations: Citation Explorer
miR-486-5p-rich extracellular vesicles derived from patients with olanzapine-induced insulin resistance negatively affect glucose-regulating function
Authors: Tu, Chuyue and Wu, Qian and Wang, Jing and Chen, Peiru and Deng, Yahui and Yu, Lixiu and Xu, Xiaojin and Fang, Xiangming and Li, Weiyong
Journal: Biochemical Pharmacology (2024): 116308
TFAIP6 facilitates hepatocellular carcinoma cell glycolysis through upregulating c-Myc/PKM2 axis
Authors: Duan, Kecai and Fang, Kunpeng and Sui, Chengjun
Journal: Heliyon (2024): e30959
CPT1A mediates the succinylation of SP5 which activates transcription of PDPK1 to promote the viability and glycolysis of prostate cancer cells
Authors: Liu, Shufeng and Chen, Xiaoguang and Zhang, Liqi and Lu, Bo
Journal: Cancer Biology \& Therapy (2024): 2329372
Prolonged control of insulin-dependent diabetes via intramuscular expression of plasmid-encoded single-strand insulin analogue
Authors: Deng, Lu and Yang, Ping and Li, Caixia and Xie, Lifang and Lu, Wanling and Zhang, Yanhan and Liu, Ming and Wang, Gang
Journal: Genes \& Diseases (2022)
Alismatis Rhizoma Triterpenes Alleviate High-Fat Diet-Induced Insulin Resistance in Skeletal Muscle of Mice
Authors: Jia, Xiao-Kang and Huang, Jin-Fang and Huang, Xiao-Qiang and Li, Xiao-Yan and Huang, Ming-Qing and Zhu, Huai-Chang and Li, Gao-Pan and Lan, Meng-Liu and Yu, Zhi-Wen and Xu, Wen and others,
Journal: Evidence-Based Complementary and Alternative Medicine (2021)

References

View all 115 references: Citation Explorer
Inhibition of the sodium glucose co-transporter-2: its beneficial action and potential combination therapy for type 2 diabetes mellitus
Authors: Chen LH, Leung PS.
Journal: Diabetes Obes Metab. (2013)
Sodium glucose co-transporter 2 (SGLT2) inhibitors: novel antidiabetic agents
Authors: Washburn WN., undefined
Journal: Expert Opin Ther Pat (2012): 483
Regulation of glucose transporter translocation in health and diabetes
Authors: Bogan JS., undefined
Journal: Annu Rev Biochem (2012): 507
New possibility in the oral glucose lowering treatment of type 2 diabetes mellitus: sodium-glucose co-transporter-2 inhibitors
Authors: Simonyi G., undefined
Journal: Orv Hetil (2012): 695
Glucose transporter type I deficiency syndrome: epilepsy phenotypes and outcomes
Authors: Pong AW, Geary BR, Engelstad KM, Natarajan A, Yang H, De Vivo DC.
Journal: Epilepsia (2012): 1503
Page updated on December 1, 2024

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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components

Measurement of 2DG uptake in differentiated 3T3-L1 adipocytes and 3T3-L1 fibroblasts. Assays were performed with Screen Quest™ Fluorimetric Glucose Uptake Assay Kit in a black wall/clear bottom cell culture Poly-D lysine plate using a Gemini (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.) 
Measurement of 2DG uptake in differentiated 3T3-L1 adipocytes and 3T3-L1 fibroblasts. Assays were performed with Screen Quest™ Fluorimetric Glucose Uptake Assay Kit in a black wall/clear bottom cell culture Poly-D lysine plate using a Gemini (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.) 
Measurement of 2DG uptake in differentiated 3T3-L1 adipocytes and 3T3-L1 fibroblasts. Assays were performed with Screen Quest™ Fluorimetric Glucose Uptake Assay Kit in a black wall/clear bottom cell culture Poly-D lysine plate using a Gemini (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.)