In Vitro Glucose Depletion Assay: A Fluorescence-Based Method to Quantify Cellular Glucose Uptake via Extracellular Glucose Depletion Measurement

Glucose, a primary cellular energy source, enters cells via cell membrane glucose transporter proteins. Glucose gets broken down in the cell cytoplasm to pyruvate, via the glycolytic pathway, generating energy through adenosine triphosphate, or ATP, for cellular metabolism.

To measure intracellular glucose uptake, begin with an adherent culture of fibroblasts in a multi-well plate. Replace the media with glucose-free media and incubate. The cells metabolize the available glucose to produce energy, attaining a glucose-starved state.

Replace the media in one set of wells with glucose-free media containing insulin, a glucose uptake stimulator, and fluorescent deoxyglucose, a glucose analog. Treat another set of wells with glucose-free media containing fluorescent deoxyglucose.

During incubation, insulin stimulates the translocation of additional glucose transporters to the plasma membrane, facilitating increased fluorescent deoxyglucose uptake, in the absence of glucose.

Deoxyglucose is phosphorylated and not metabolized further by cells. Concurrently, the extracellular concentration of fluorescent deoxyglucose decreases.

Post-incubation, collect the media containing unutilized fluorescent deoxyglucose. Treat the cells with a lysis buffer. The detergent in the buffer forms micelles and solubilizes the cellular membranes, causing intracellular content release.

Using a microplate reader, measure the fluorescence of the cell homogenate containing intracellular fluorescent deoxyglucose and media containing unutilized extracellular fluorescent deoxyglucose.

Insulin-stimulated fibroblasts exhibit increased intracellular fluorescent deoxyglucose uptake and corresponding decreased extracellular fluorescent deoxyglucose compared to non-stimulated cells.

Use 8 replicates for each simulation condition. Therefore, prepare 1 milliliter for each simulation condition.

Use 7 simulation conditions without insulin: FD-glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 milligram per milliliter in one 96-well plate.

Use 7 stimulation conditions with insulin: FD-glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 microgram per milliliter in another 96-well plate.

To prepare stimulation conditions, dilute 1 microliter of 5 milligrams per milliliter FD-glucose working solution and 999 microliters of glucose-free DMEM to obtain 1 milliliter of working stock solution.

Using this working stock solution, prepare 1:2000, 1:5000, 1:10000, 1:25000, 1:50000, and 1:100,000 dilutions of FD-glucose to obtain 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter in 2-milliliter tubes immediately before experiments in a biosafety cabinet without lights. Add 1 microliter of insulin to each of these conditions.

After 40 minutes of incubation, decant the glucose-free DMEM from each well in 96-well plates, and absorb the remaining fluid with sterile paper towels. Add 100 microliters each from the various treatment conditions described before, to wells along one column, and 100 microliters from the same treatment condition to the wells along the row in both 96-well plates. Label the plates that utilized the conditions with and without insulin.

Add glucose-free DMEM alone to the control wells. Incubate the plate for 40 minutes in a cell culture incubator in the dark.

After the cells have been stimulated for 40 minutes, transfer the stimulation media from both the plates into new 96-well plates, maintaining the same experimental layout. Wash the cells with PBS. Remove PBS and decant any remaining solution on sterile paper towels.

Add a protease inhibitor to the radioimmunoprecipitation assay buffer to protect proteins. Place plates containing radioimmunoprecipitation assay in a shaker for 30 minutes.

Using a microplate reader, measure fluorescence at excitation and emission wavelengths of 485 and 535 nanometers respectively, first, in the medium containing extracellular FD-glucose, and then, the plate with radioimmunoprecipitation assay-lysed cells at the end of 30 minutes incubation.