Cell Meter™ Glucose Uptake Imaging Kit *Red Fluorescence*

Thaw all the components at room temperature before starting the experiment.

1. Prepare cells with your test compounds.

2. Add Glutite™ Red 670 dye working solution.

3. Incubate cells at 37 °C for 30 to 60 minutes.

4. Remove Glutite™ Red 670 dye working solution.

5. Wash cells with PBS.

6. Analyze cells using a fluorescence microscope with a Cy5 filter.

1. Grow 3T3-L1 fibroblasts 2 days post-confluence in a 75 cm flask using DMEM supplemented with 10% FBS.

2. To initiate differentiation of 3T3-L1 cells, remove the medium and add DMEM supplemented with 10% FBS, 0.83 µM insulin, 0.25 µM dexamethasone, and 0.25 mM isobutylmethylxanthine. Incubate for 2 days.

3. Remove the medium and add DMEM supplemented with 10% FBS and 0.83 µM insulin. Incubate for 2 days.

4. Remove the medium and add DMEM supplemented with 10% FBS. Incubate for 3-5 days.

5. Differentiated cells (at least 95% of which showed an adipocyte phenotype by the accumulation of lipid droplets) were used on days 8 to 12 after induction of differentiation.

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

1. Add 100 μL of DMSO into Glutite™ Red 670 vial (Component A) to make a stock solution.

   Note: Any unused Glutite™ Red 670 stock solution should be divided into single-use aliquots and stored at ≤ -20 º C. Protect from light and avoid repeated freeze-thaw cycles.

1. Prepare the working solution by adding 100 μL of Glutite™ Red 670 stock solution into 10 mL of Assay Buffer (Component B).

   Note: Protect the working solution from light by covering it with foil or placing it in the dark.

   Note: For best results, this solution should be used within a few hours of its preparation.

   Note: 10 mL of working solution is enough for 100 tests.

1. Add 20 mL of 5X KRPH buffer (Component D) to 80 mL of deionized water.

   Note: 50 mL volume of 1X KRPH buffer is enough for approximately one 96-well plate. Prepare the needed volume proportionally. Store any unused 1X KRPH buffer at 4ºC or -20 ºC.

The following protocol serves as guidelines to culture 3T3-L1adipocytes for Glutite™ Red 670 uptake in a 96-well plate, and should be modified according to your specific needs.

1. Plate the entire 75 cm flask of 3T3-L1 adipocytes, 100 µL/well, in a 96-well black wall/clear bottom cell culture Poly-D lysine plate for 4-6 hours before the 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 serum-free medium. Incubate the cells at 37 ºC, 5% CO₂ 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 1X KRPH buffer.

4. Add 90 µL/well of Assay Buffer (Component B) and incubate the cells at 37 ºC, 5% CO₂ incubator for 1 hour.

5. Treat cells with insulin, without insulin, or with the test compound for 2 hours. Add 10 µL/well of the 10X insulin solution for a final concentration of 1 µM or add 10 µL/well of the 10X test compound solution. Additionally, add 10 µL of insulin vehicle buffer or compound vehicle buffer to the untreated wells as control, and incubate at 37 ºC, 5% CO₂ for 60 minutes.

6. For the glucose uptake inhibition study, add 10X Phloretin to a final concentration of 200 µM or test inhibitors, and incubate at 37 ºC, 5% CO₂ for 60 minutes.

   Note: It is recommended to add 10 µL of inhibitor vehicle buffer to both the insulin-treated and untreated wells as a control.

7. Add 100 µL/well of Glutite™ Red 670 dye working solution, and incubate for 60 minutes.

   Note: Optimal incubation time will need to be determined for each cell line and experiment

8. Remove Glutite™ Red 670 dye staining solution without disturbing the cells.

9. Wash the cells with PBS twice.

10. Keep the cells in 100 µL/well (96-well plate) of Assay Buffer (Component B).

11. Monitor the fluorescence signal using a fluorescence microscope with a Cy5 filter set.