Cell Meter™ Glucose Uptake Imaging Kit *Red Fluorescence*
|Shipping||Standard overnight for United States, inquire for international|
|Extinction coefficient (cm -1 M -1)||250000|
|Quantum yield||0.271, 0.42|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Storage||Freeze (< -15 °C); Minimize light exposure|
Extinction coefficient (cm -1 M -1)
|Excitation||Cy5 Filter Set|
|Emission||Cy5 Filter Set|
|Recommended plate||Black wall/clear bottom|
AT A GLANCE
Thaw all the components at room temperature before starting the experiment.
Prepare cells with your test compounds.
Add Glutite™ Red 670 dye working solution.
Incubate cells at 37 °C for 30 to 60 minutes.
Remove Glutite™ Red 670 dye working solution.
Wash cells with PBS.
Analyze cells using a fluorescence microscope with a Cy5 filter.
Grow 3T3-L1 fibroblasts 2 days post-confluence in a 75 cm flask using DMEM supplemented with 10% FBS.
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.
Remove the medium and add DMEM supplemented with 10% FBS and 0.83 µM insulin. Incubate for 2 days.
Remove the medium and add DMEM supplemented with 10% FBS. Incubate for 3-5 days.
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.
PREPARATION OF STOCK SOLUTIONS
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
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.
PREPARATION OF WORKING SOLUTION
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.
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.
SAMPLE EXPERIMENTAL PROTOCOL
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.
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.
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.
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.
Add 90 µL/well of Assay Buffer (Component B) and incubate the cells at 37 ºC, 5% CO2 incubator for 1 hour.
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.
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.
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
Remove Glutite™ Red 670 dye staining solution without disturbing the cells.
Wash the cells with PBS twice.
Keep the cells in 100 µL/well (96-well plate) of Assay Buffer (Component B).
Monitor the fluorescence signal using a fluorescence microscope with a Cy5 filter set.
|Extinction coefficient (cm -1 M -1)||250000|
|Quantum yield||0.271, 0.42|
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