Encyclopedia of Experiments: Cancer Research
A subscription to JoVE is required to view this content.
Transcript
- Extracellular flux assay helps study cell metabolism by measuring the two major energy pathways-- glycolysis and mitochondrial respiration. During glycolysis, cells metabolize glucose and secrete lactate into the extracellular space along with protons, determined by measuring the Extracellular Acidification Rate or ECAR. During mitochondrial respiration, cells utilize oxygen for ATP production, measured by determining the Oxygen Consumption Rate or OCR.
Cancer cells exhibit high glucose uptake as they alter their metabolic rate to adapt and maintain unregulated cellular proliferation. To analyze metabolism in cancer cells, first assemble a flux analysis microplate containing adherent cancer cell monolayer and a hydrated sensor cartridge. Transfer the plate assembly into a flux analyzer. Within the analyzer, the sensor probes descend to create a transient microchamber. To measure extracellular flux, the injection port introduces test compounds into the microplate at preset time points.
Based on cell metabolism, the concentrations of oxygen available and protons released in the microchamber fluctuates. Two fluorophores embedded at the base of the cartridge simultaneously senses and measures the oxygen and proton levels, indicative of mitochondrial respiration and glycolysis, respectively. In the following protocol, we will demonstrate an extracellular flux assay to measure the metabolic activity of primary leukemic cells.
- Meanwhile, to prepare the plates for extracellular flux analysis, add 12.5 microliters of cell-adhesive solution into each well of two 8-well extracellular flux analyzer plates. After 20 minutes, aspirate the cell adhesive and wash each well two times with 200 microliters of sterile water per wash.
After the second wash, leave the plates in the hood until the wells are dry, and place the sensor cartridge upside down on the lab bench. Separate the utility plate and the sensor cartridge of the flux analyzer, and fill each well of the utility plate with 200 microliters of calibrant and each moat around the outside of the wells with 400 microliters of calibrant.
Return the sensor cartridge to the utility plate that now contains the calibrant, and place the cartridge assembly in a humidified, 37 degrees Celsius incubator without CO2 overnight. Then turn on the extracellular flux analyzer and let it warm to 37 degrees Celsius overnight.
The next morning, transfer the cells from the flask to 50 milliliter conical tubes for centrifugation, and resuspend the pellets in 1 milliliter of the appropriate experimental medium for counting. Resuspend the cells at 4 times 10 to the 6 cells in 400 microliters of experimental medium concentration and add 50 microliters of cells into wells B through G of the flux analyzer plate, and 180 microliters of the experimental medium into wells A and H as the background control wells.
After centrifugation, slowly and carefully add 130 microliters of the experimental medium to wells B through G, and visually confirm stable adherence of the cells to the bottom of each well under the microscope. Then return the flux analysis plate to the humidified, 37 degrees Celsius incubator without CO2 for 30 minutes.
20 minutes before the end of the incubation, load the compounds into the appropriate injector ports of the cartridge according to the experimental protocol, as indicated in the table. Then set up the appropriate extracellular flux analysis program. Start the program, and replace the calibrant plate with the assay plate when prompted.
