Glucose Uptake Assay Kit-Green

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

The comparison of the Glucose Uptake Probe Series and the existing method(2-NBDG) is as below.

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.

[Flow cytometry] Example procedure: 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

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

Experimental Examole: Induction of Ferroptosis by Erastin

Erastin is a known inducer of ferroptosis. By inhibiting the cystine transporter (xCT), erastin inhibits the uptake of cystine. Cystine is the raw material for GSH. Therefore, Erastin ultimately decreases the amount of GSH. Decreased GSH then results in lipid peroxide accumulation and induction of ferroptosis.
The following experimental examples show changes in each aforementioned index as a consequence of erastin stimulation. Measurements are made using Dojindo reagents.

Using erastin-treated A549 cells, we measured intracellular Fe²⁺, ROS, lipid peroxide, glutathione, glutamate release into the extracellular space, and cystine uptake. As a result, inhibition of xCT by elastin was observed and also the release of glutamate and uptake of cystine were decreased. Furthermore, elastin treatment decreased intracellular glutathione while it increased intracellular Fe²⁺ , ROS, and lipid peroxides.

Cell Line: A549
Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h