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Glucose Uptake Assay Kit-Green

Item # Unit Size
UP02-10
1 set

For Research Use Only Products

- Highly sensitive and simple measurement of glucose uptake capacity
- Applicable for a plate reader
- Reduces dye leakage after staining

Contents: 1 set


The general number of usable assays per 1 set


Storage Condition: Store at 0-5oC
Shipping Condition: Ambient Temperature
MSDS

Nutrient metabolism is necessary for energy production in cells and regulates various cellular functions, including gene expression. Glucose is one of the key substrates for the generation of ATP and to sustain cellular homeostasis. Thus, glucose metabolism has been the subject of intense investigations. In cancer research, tumor cells enhance glucose uptake and consumption for their growth and proliferation. Therefore, elevated glucose uptake is a marker of tumors, and glucose transporters are important targets in cancer treatment. One common method for evaluating the glucose uptake ability of cells uses radioisotope-labeled glucose. Although this method has been used for many years, it requires special handling facilities and disposal of radioactive materials. The enzyme cycling method using 2-deoxy-D-glucose, which enables colorimetric and fluorometric plate assays, cannot be applied to cell imaging and flow cytometry. Recently, 2-NBDG, a fluorescently labeled glucose analog, has been used widely to detect cellular glucose uptake by fluorescence imaging and flow cytometry1). However, the sensitivity of this method is poor because of the low fluorescence intensity of 2-NBDG. To resolve these limitations, a novel fluorescent probe, Glucose Uptake Probe-Green, was developed. This probe emits strong green fluorescence (λex = 507 nm, λem = 518 nm), allowing highly sensitive detection of cellular glucose uptake by fluorescence imaging, flow cytometry or microplate assay. The WI Solution in this kit enhances cellular retention of the probe to give more reliable data.

The use of a high-fluorescence intensity dye enables higher sensitivity measurements in a shorter time than the conventional method (2-NBDG).

1. High-sensitivity determination

Uses a fluorescent dye with superior brightness, which is different from the fluorescence intensity of 2-NBDG, which decreases in water.



Deteciton conditions
Cells: A549 cells
Detection system: Fluorescence microscope
Filter set: GFP (Ex: 470/40 nm, Em: 525/50 nm)

2. Quick process

Significantly reduced measurement time despite using the same process as 2-NBDG.



The operation is very simple, requiring only three steps: pretreatment, staining (incorporation), and washing.



3. Plate reader compatibility

Supports plate reader measurements that are difficult to perform with 2-NBDG.



Deteciton conditions
Cells: A549 cells
Ex: 488 nm, Em: 520 nm

4. Reduces dye leakage after staining

The included WI Solution suppresses reagent leakage and ensures stable data acquisition.

When cells are washed with HBSS



When cells are washed with WI Solution



Deteciton conditions
Cells: A549 cells
Detection system: Fluorescence microscope
Filter set: GFP (Ex: 470/40 nm, Em: 525/50 nm)

Comparison with existing method

Glucose Uptake Probe-Green is compatible with fluorescence microscopes and flow cytometers as well as 2-NBDG. The excitation wavelength is more suitable for 488 nm excitation lasers and filter sets compared with 2-NBDG.



* This is the result of a study using A549 cells, and the leakage time varies depending on the cell type

Difference from Glucose Assay Kit

Difference between Glucose Uptake Assay Kit-Green and Glucose Assay Kit-WST (product code: G264)

1. Glucose Uptake Assay Kit-WST can quantify glucose consumption in cell supernatant. On the other hand, Glucose Uptake Assay Kit-Green cannot quantify glucose.

2. Glucose Uptake Assay Kit-Green can measure the difference of glucose uptake capacity in a short time, while Glucose Assay Kit-WST cannot measure the change of glucose level in a short time.

The difference between the Glucose Assay Kit-WST and this kit will be explained using the following experimental example.

The difference between the Glucose Assay Kit-WST and this kit will be explained using the following experimental example.
Example of experiment: Measurement of glucose consumption and uptake capacity of HepG2 treated with glucose uptake inhibitor (Cytochalasin B).
Flowchart of the experiment and experimental results



Experimental example: Inhibition of glucose uptake by Cytochalasin B

Using this kit, we were able to detect the inhibition of glucose uptake by the glucose transporter inhibitor Cytochalasin B in HepG2 cells with high sensitivity.

Fluorescence microscope observation



(scale bar: 50 μm)

Measurement conditions
Cells: HepG2
Medium: MEM (5.5 mmol/l Glucose)
Culture conditions: 5 µmol/l Cytochalasin B/MEM (5.5 mmol/l Glucose, 10% FBS), 37 °C, 24 h
Staining conditions: Glucose Uptake Probe/DMEM (0 mol/l Glucose) diluted by 500-fold, 37 °C, 15 min
Detection system: Fluorescence microscope
Filter set: GFP (Ex: 470/40 nm, Em: 525/50 nm)

Plate reader detection



Ex: 488 nm, Em: 520 nm

Experimental example: Enhanced glucose uptake by insulin

Using this kit, we were able to measure enhanced insulin-induced glucose uptake in adipocytes with high sensitivity.



(scale bar: 50 µm)

Measurement conditions
Cells: mouse adipocytes
Medium: DMEM (5.5 mmol/l Glucose)
Culture conditions: 1 µmol/l Insulin/DMEM (5.5 mmol/l Glucose), 37 °C, 15 min
Staining conditions: ×500 Glucose Uptake Probe/DMEM (0 mol/l Glucose), 37 °C, 15 min
Detection system: Fluorescence microscope
Filter set: GFP (Ex: 470/40 nm, Em: 525/50 nm)
Scale bar: 50 μm

Plate reader detection



Detection conditions
Ex: 488 nm, Em: 520 nm

* It is difficult to make adipocyte evenly on the wells, so there may be some error in the data.

Experimental procedure
1. Adipocytes were seeded onto ibi 96-well plates and cultured overnight.
2. The cells were washed twice with glucose-free medium DMEM, and then glucose-free medium was added.
3. The cells were incubated at 37 °C for 15 minutes.
4. Probe solution (500-fold dilution) in glucose-free medium was added, and the cells were incubated at 37 °C for 15 minutes.
5. Wash the cells three times with WI Solution (1x) chilled to 4 °C and add WI Solution (4 °C).
6. The cells were observed using a fluorescence microscope and plate reader.

Experimental example: Comparing the glucose uptake capacity of progenitor adipocytes and adipocytes

Using this kit, we were able to detect and quantify the difference in glucose uptake capacity between progenitor adipocytes and adipocytes with high sensitivity.



(scale bar: 50 µm)

Measurement conditions
Cells: preadipocyte, adipocyte
Medium: DMEM (5.5 mmol/l Glucose, 10% FBS)
Staining conditions: Glucose Uptake Probe/DMEM (0 mol/l Glucose) diluted by 500-fold, 37 °C, 15 min
Detection system: Fluorescence microscope
Filter set: GFP (Ex: 470/40 nm, Em: 525/50 nm)

Plate reader detection



Detection conditions
Ex: 488 nm, Em: 520 nm

* It is difficult to make adipocyte evenly on the wells, so there may be some error in the data.

Experimental procedure
1. Progenitor adipocytes and adipocytes were seeded onto ibi 96-well plates and cultured overnight.
2. The cells were washed twice with glucose-free medium DMEM, and then glucose-free medium was added.
3. The cells were incubated at 37 °C for 15 minutes.
4. Probe solution (500-fold dilution) in glucose-free medium was added, and the cells were incubated at 37 °C for 15 minutes.
5. Wash the cells three times with WI Solution (1x) chilled to 4 °C and add WI Solution (4 °C).
6. The cells were observed using a fluorescence microscope and plate reader.
Experimental Examples:

Co-staining of Glucose uptake probe-Green and markers for immunostaining

1. Wash the cells with PBS x 2
2. Stain zombie yellow
3. Incubate for 30 min at 4°C
4. Wash the cells with RPMI (1% FCS) x 2
5. Stain cell surface antibody
6. Incubate for 30 min at 4°C
7. Wash the cells with RPMI-No glucose x 2
8. Add pre-warmed RPMI (37°C), divide into five

Sample GLUP-G Media GLUT inhibitor
1 - RPMI-No glucose -
2 + RPMI-No glucose -
3 + RPMI with glucose -
4 + RPMI-No glucose +
5 + RPMI-No glucose +

9. Incubate for 15 min at 37 °C in a CO2 incubator
10. Centrifuge at 1,500 rpm for 5 min at r.t., remove the supernatant
11. Add pre-warmed Glucose uptake probe-Green (+/- inhibitor) for 15 min at 37 °C in a CO incubator
12. Wash with ice-cooled WI solution x 2
13. FCM analysis
Q. Does Glucose Uptake Probe-Green have selectivity for the type of glucose transporter?

A. No. Glucose Uptake Probe-Green is thought to be taken up by cells via multiple glucose transporters. However, there are no detailed data on the selectivity and affinity of this probe for glucose transporters.


Q. What cell types have you used Glucose Uptake Probe-Green on?

A. We have used the probe in the following cell types.
Cell types Dilution factor of probe stock solution Staining time
Human alveolar basal epithelial adenocarcinoma cells A549 ×500 15 min
Human hepatoma-derived cells HepG2 ×500 15 min
Progenitor adipocytes Preadipocytes (3T3-L1) ×500 15 min
Adipocytes Adipocytes (3T3-L1) ×500 15 min
Malignant melanoma MO5 ×500 15 min
Mouse myoblasts C2C12 ×500 5 min
Astrocyte-ma U-251 MG ×500 15 min
Human cervical cancer-derived cells HeLa ×500 15 min
Lewis lung cancer-derived cells 3LL ×50000 15 min
T cells CD4+ T cell ×50, ×500 15 min
Macrophage-like cells J774.1 ×500 15 min
Nematode N2 ×500 90 min

Q. Is it possible to fix the cells after staining with Glucose Uptake Probe-Green?

A. The glucose moiety may undergo phosphorylation by hexokinase owing to its structure, but no further metabolism is expected to occur.


Q. After Glucose Uptake Probe-Green is incorporated into cells, can the cells be fixed?

A. No. Glucose Uptake Probe-Green leaks from inside the cells after fixation.


Q. What type of microplate should be used for cell culture?

A. Please use a black microplate for cell culture.


Q. Can I save the Probe working solution?

A. No, the Probe working solution cannot be stored. The Probe Stock solution can be stored -20oC for 1 month.


Q. What should I do if the sensitivity of measurement using Glucose Uptake Probe-Green is low?

A. Please consider the probe concentration (×250 to ×1,000) and staining time (15 minutes to 1 hour) as initial considerations.


Q. What should I do if the fluorescence background is high in the measurement using Glucose Uptake Probe-Green?

A. Please optimize the staining time and dilution factor of the Probe with reference to the following ranges.

Probe dilution concentration: ×250 to ×1,000

Staining time: 15 to 60 minutes.

There is a possibility that Glucose Uptake Probe-Green remains in the extracellular region. Therefore, please wash the cells again with WI Solution.


Q. Does Glucose Uptake Prob-Green have any cytotoxicity?

A. There was no cytotoxicity. Using our product Cell Counting Kit-8 (product code CK04), we measured the cytotoxicity of this probe in A549 cells.


Q. How long is the probe retained in the cells after washing with WI Solution?

A. The Probe is retained in the cells for about 1 hour at room temperature in WI Solution. Please note that the retention time may vary depending on the cell type.


Q. Can the Glucose Uptake Assay Kit-Green be used to quantify glucose in the culture medium or in cells?

A. No, please use our product Glucose Assay Kit-WST (product code G264).


Q. Is it possible to quantify Glucose Uptake Probe-Green taken up into cells?

A. No, this kit measures the fluorescence intensity of the probe taken up into the cell as an indication of the glucose uptake capacity of the cell.