Glucose is a principal carbon source for ATP production and biosynthesis in the multiplication of mammalian cells by either tricarboxylic acid (TCA) or glycolytic cycle pathways. These pathways are firmly regulated and their disruption results in several metabolic diseases. Cancer cells are characterized by increased glucose uptake in order to support their high proliferation rate. Glucose uptake has been extensively studied by a non-radioactive fluorescence assay that employs a fluorescently labeled glucose derivative, 2-NBD-Glucose. In spite of its applicability, this conjugate shows signs of photo bleaching and decreased fluorescence when used at low concentrations (<400 µM).
BioVision has developed innovative bright green fluorescent conjugates (GluTracker™ and AGTracker™). Quite similar to glucose and 2-NBD-Glucose, GluTracker™ and AGTracker™ can be taken up by cells through glucose transporters. Furthermore, these glucose analogs cannot be fully metabolized by cells, and thus build up inside the cells to generate strong fluorescent signals.
Fluorescence produced by these fluorescent glucose analogs is proportional to the glucose uptake by the cells, and can be used to measure glucose uptake using flow cytometry and fluorescence microscopy. AGTracker™ and GluTracker™ serve as valuable tools for the study of anhydroglucitol and glucose uptake.
Experimental Flow Chart
Structure for GluTracker™ and AGTracker™
Figure 1. (A) Structure of GluTracker™ (B) Structure of AGTracker™
Cellular Glucose Uptake
Figure 2. Glucose Uptake in cells can be carried out in two ways: Na + dependent transporter, also called: SGLT (an active process which requires ATP) and Na + independent transport, also called GLUT. Phloretin, a dihydrochalcone, is a natural phenol capable of inhibiting cellular glucose active transport.
GluTracker™ and AGTracker™ are Used to Detect Glucose Uptake
Figure 3. Time-course study for GluTracker™ and 1,5-AGTracker™ Uptake Assays. 50 µM GluTracker™ (A) or 50 µM AGTracker™ (B) are used to measure the glucose uptake in Jurkat cells for 0 (Purple, filled), 5 (Green), and 60 minutes (Orange).
GluTracker™ and AGTracker™ are Robust Tools for Glucose Uptake
Figure 4. Dose-response study for GluTracker™ and AGTracker™ Uptake Assays. (A) The glucose uptake in Jurkat Cells is measured by using different concentration of GluTracker™: 0(Purple filled), 12.5 µM (Green), and 100 µM (Red). (B) The glucose uptake in Jurkat Cells is measured by using different concentration of AGTracker™: 0 (Purple filled), 12.5 µM (Green), and 100 µM (Red).
Phloretin Inhibits/Impedes Glucose Uptake in Jurkat Cells
Figure 5. Jurkat cells were treated with or without 1X phloretin for 45 minutes. (A) After treatment, cells were washed and incubated with 50 µM GluTracker™ Reagent, GluTracker™ Enhancer, and 1X phloretin for another 30 minutes, according to protocol. Black: negative control cells; Orange: in the presence of phloretin; Blue: without phloretin. The percentage of inhibition by phloretin is 45%. (B) After treatment, cells were washed and incubated with 50 µM AGTracker reagent and the same concentration of phloretin for another 30 minutes, according to protocol. Black: negative control cells; Orange: in the presence of phloretin; Blue: without phloretin. The percentage of Inhibition by phloretin is 50%.
Visualization of Glucose and 1,5-Anhydroglucitol Uptake by Using GluTracker™ and AGTracker™
Figure 6. Glucose uptake was evaluated in HeLa cells using BioVision’s GluTracker™ Assay Kit (A) or BioVision’s AGTracker™ Assay Kit (B). Cells were treated with or without 1X phloretin for 45 minutes. After treatment, cells were washed and incubated with either 50 µM GluTracker™ or 50 µM AGTracker™ for 30 minutes, according to kit protocol. Images were taken using fluorescence microscope (Top Panel: treat with phloretin; Middle Panel and Bottom Panel: without 1X phloretin). Top and Middle Panels: 20X Objective lens; Bottom Panel: 60X Objective lens. HeLa cells showed the uptake in the cytoplasm.
Key Advantages of GluTracker™ and AGTracker™
- Versatile: fluorescence microscopy and flow cytometry
- Simple protocol, sensitive, bright fluorescence signal and reproducible
- Extensively applicable for various cell types: Jurkat, HeLa, and MCF-7, and so on
- Staining of 1,5-anhydroglucitol and glucose uptake
- Analysis of glucose metabolism and cell signaling in different cell types
- Screening of anti-diabetic compounds
- Measurement of 1,5-anhydroglucitol and glucose uptake in response to insulin, cytokines, growth factors, and so on
BioVision’s AGTracker™ (for 1,5-AG) and GluTracker™ (for Glucose) Uptake Assays are useful tools for drug discovery and identifying potential modulators in glucose-dependent pathways.
BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.
BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.
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