Measurement of Extracellular Fluorescent Glucose Analog Depletion: A Surrogate Measurement Technique to Assess Glucose Uptake in Mouse Organs Ex Vivo
Trascrizione
Incubate the harvested tissues or organs in a 6-well plate containing PBS for one minute. Handle each tissue or organ in a separate well. After one minute, place each tissue on a sterile paper towel to absorb PBS.
Transfer tissues or organs in a separate 6-well plate containing 4,000 microliters of glucose-free DMEM, and incubate for two minutes. After two minutes, remove and transfer the tissues into wells of a 6-well plate containing 0.29 millimolar FD-glucose working solution. Incubate the 6-well plates containing tissues in FD-glucose working solution at 37 degrees Celsius.
Collect 100 microliters of FD-glucose working solution from each well after 0, 10, 20, 30, 40, 60, 90, and 120 minutes of incubation, and transfer collected 100 microliters of FD-glucose working solution into 96-well plates to analyze the kinetics of extracellular FD-glucose depletion. Shake before and after collection.
Measure the fluorescence at excitation and emission wavelengths of 485 and 535 nanometer respectively using a microplate reader.
