This method can help answer key questions regarding metabolic pathway setup in leukemia cells. The main advantage of this technique is that it allows measurement of the metabolism of primary leukemia cells in real time in live cells. Begin by diluting a bone marrow sample obtained from a leukemia patient in PBS at a one to one ratio.
Carefully, layer six milliliters of the diluted bone marrow sample over six milliliters of freshly prepared density gradient medium in a 15 milliliter conical tube and separate the cells by density gradient centrifugation. Use a Pasteur pipet to carefully transfer the interface layer of mononuclear cells to a new 50 milliliter conical tube containing five milliliters of PBS for a centrifugation wash. Then, resuspend the mononuclear cell pellet in two milliliters of sterile PBS for counting.
Dilute the cells to a three times 10 to the seven cells per milliliter concentration. And add one milliliter of cells to each of two T75 flasks containing 20 milliliters of RPMI medium for a 16 to 24 hour incubation at 37 degrees Celsius and 5%CO2. Meanwhile to prepare the plates for extracellular flux analysis, add 12.5 microliters of cell adhesive solution into each well of two, eight 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 one milliliter of the appropriate experimental medium for counting.
Resuspend the cells at four times 10 to the six 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. In a glycolysis stress test, only basal medium is used so that the cells are deprived of nutrients.
The first parameter obtained is the basal acidification which should reflect the amount of glucose stored in cells. After the first injection, the extracellular acidification rate is increased as the cells utilize the glucose and can ferment the glucose to lactate. The oligomycin A in the second injection inhibits ATP synthase and thus directs the cells to produce ATP mainly by a glycolysis causing further elevation of the extracellular acidification rate.
While the injection of 2-Deoxy-D-glucose completely inhibits glycolysis and the extracellular acidification rate drops. In the cell mito stress test, a medium supplemented with glutamine and glucose is used so that the cells are not deprived of all nutrients. The basal respiration parameter reflects their basal metabolic state.
After the first injection with oligomycin A, the cells inhibit mitochondrial respiration and switch to glycolysis which is represented as a decrease in the oxygen consumption rate. The second and third injections of FCCP however, uncouple the ATP production from the respiration so that the cells now consume oxygen at a maximal rate. And the oxygen consumption rate rises to its highest value.
The last injection of the rotenone and antimycin A mixture completely inhibits the mitochondrial respiration decreasing the oxygen consumption rate to almost zero. While attempting this procedure, it's important to assess the percentage of blasts in the sample to ensure that the metabolic parameters of the leukemiablasts only are measured. When implementing this method, a careful optimization has to be applied to cultivation conditions and data normalization.
