Metabolic changes in hematopoietic stem and progenitor cells allow them to transition from their quiescent state to a differentiation state to sustain demands of blood formation. Alteration in metabolic plasticity of hematopoietic stem and progenitor cells leads to hematologic disorders. Our protocol will help measure the metabolic regulation and the health of hematopoietic stem and progenitor cells during blood development and diseases.
Analysis of the mitochondrial respiration and glycolysis of hematopoietic stem and progenitor cells is difficult as they are fragile and rare population. Our optimized protocol allows the isolation of a higher amount of hematopoietic stem and progenitor cells from murine bone marrow, improves their viability during incubation, facilitates extracellular flux analysis of non-adherent HSPCs, and provide optimized injection parameters for drug targeting oxidative phosphorylation as well as glycolytic pathways. To begin this procedure, fill the wells of the utility plate and the chambers around the wells with sterile water.
Submerge the sensor cartridge into the utility plate making sure that the sensors are completely covered by water. Then place the cartridge submerged in the utility plate in a non CO2 incubator at 37 degrees Celsius to incubate overnight. In a class II biosafety cabinet, add 40 microliters of commercially available 0.1%PLL solution to each well of the assay plate.
Cover the assay plate with the lid provided in the kit and incubate the closed plate at room temperature under the hood for one hour. After this, use a sterile vacuum-based aspirator to remove the excess solution and air dry the plate under the hood. To harvest mononucleated murine bone marrow cells, add five milliliters of density gradient medium to a 15 milliliter conical tube.
Slowly add five milliliters of the bone marrow cell suspension making sure that the cells remain as a layer above the density gradient medium. Centrifuge at 500 times g and at room temperature for 30 minutes making sure that the centrifuge is at the lowest possible acceleration. Harvest the middle interface of mononucleated cells into a fresh 15 milliliter tube.
Then wash the cells with five milliliters of 1X PBS containing 2%FBS. Centrifuge at 500 times g and at four degrees Celsius for five minutes. Remove the supernatant and resuspend the cells in 300 microliters of 1X PBS containing 2%FBS.
Next, aliquot 10 microliters of the cell suspension for unstained or single-colored control in a FACS tube. To harvest LSK HSPCs from mononucleated murine bone marrow cells, add 15 microliters of biotin antibody cocktail to 300 microliters of mononucleated bone marrow cells. To harvest LSK HSPCs from mononucleated murine bone marrow cells, add 15 microliters of biotin antibody cocktail to 300 microliters of mononucleated bone marrow cells.
Place the tubes in an ice bucket positioned on a shaker in a refrigerator to incubate at four degrees Celsius with agitation for 30 minutes. Then add 10 milliliters of pre-chilled 1X PBS containing 2%FBS to the cells. Centrifuge at 500 times g and at four degrees Celsius for five minutes.
Discard the supernatant and resuspend the cell pellet in 400 microliters of 1X PBS containing 2%FBS. Briefly vortex the anti-biotin microbeads before use. Add 80 microliters of the microbeads to each sample and mix well.
Incubate for an additional 20 minutes at four degrees Celsius with agitation. After this, add 10 milliliters of pre-chilled PBS containing 2%FBS to the cells. Centrifuge the tube at 500 times g and at four degrees Celsius for five minutes.
Discard the supernatant and resuspend the cell pellet in one milliliter of PBS containing 2%FBS. Store the cell suspension at four degrees Celsius while setting up the magnetic separation unit. Place the column in a magnetic field for the magnetic assisted cell sorting separator at four degrees Celsius.
Prepare the column for magnetic separation by rinsing it with three milliliters of 1X PBS containing 2%FBS under gravity flow at four degrees Celsius. Add the cell suspension to the pre-wet column at four degrees Celsius. Allow all the cells to pass through the column at four degrees Celsius and collect the effluent in a 15 milliliter conical tube.
Next, wash the column with three milliliters of 1X PBS and 2%FBS at four degrees Celsius. Repeat this wash three times. Collect the flow through and keep it at four degrees Celsius.
Centrifuge the conical tube containing the flow through at 500 times g and at four degrees Celsius for five minutes. Discard the supernatant and resuspend the cells in 0.5 milliliters of 1X PBS with 2%FBS and transfer the suspension to a FACS tube. Then add 24 microliters of the LSK antibody cocktail to each 10 million cells.
Cover the tube with foil and incubate in the dark at four degrees Celsius with agitation for one hour. After this, add three milliliters of 1X PBS with 2%FBS to the FACS tube. Centrifuge at 500 times g and at four degrees Celsius for five minutes.
Discard the supernatant and resuspend the antibody-labeled cells in one milliliter of 1X PBS containing 2%FBS. Just before sorting, add one microliter of one milligram per milliliter propidium iodide to the cell suspension and use a 40 micrometer strainer to filter the contents of the FACS tube. First, centrifuge the collected LSK cells at 500 times g and at room temperature for five minutes.
Discard the supernatant and resuspend the cells in complete media to a final concentration of at least 70, 000 cells per 40 microliters. Seed 40 microliters of this cell suspension into the wells of a PLL coated eight-well plate making sure to leave all of the corner wells empty. Centrifuge the plate at 450 times g and at room temperature for one minute.
Use an inverted microscope to ensure the cells are attached to the bottom of the wells. After this, add 135 microliters of complete media to the cells in each well bringing the final volume of each well to 175 microliters. Add 175 microliters of complete media to the two corners of the plate as blanks.
Incubate the cells in a non CO2 incubator at 37 degrees Celsius for two hours. Then set up a program for the analyzer to add drugs to each well of the plate as outlined in table two and table three of the text protocol. For the mitochondrial stress test, retrieve the sensor cartridge in the utility plate from the incubator such that each well has a final concentration of two micromolar oligomycin, 1.5 micromolar FCCP, and 0.5 micromolar of either rotenone and anti-mycin A as needed.
Remove the lid from the sensor cartridge and place it on the instrument tray. Begin the calibration process which will take 20 minutes. When calibration is complete, remove the utility plate and substitute it with the assay plate containing the LSK cells.
Press continue to start the previously set program. When the program is complete, retrieve the data and analyze it. In this study, a maximum amount of viable murine LSK HSPCs are isolated and their glycolysis and mitochondrial metabolism is measured with an extracellular flux analyzer.
Although extracellular flux analysis is traditionally used for adherent cells, this method makes LSK HSPCs adherent to PLL coated wells which allows for measurement of the extracellular flux and thus metabolic health of LSK HSPCs. In order to measure the mitochondrial respiration through the oxygen consumption rate and glycolysis through the extracellular acidification rate in basal and stress conditions, drugs that interfere with glycolysis and mitochondrial respiration are injected sequentially. As expected, the basal level of the extracellular acidification rate is higher for LSK HSPCs cultured in glucose and pyruvate positive media compared to those cultured in negative media.
The injection with glucose did not change the extracellular acidification rate for the cells cultured in the positive media, but it boosted the glycolysis of cells in negative media. However, the basal level of these cells still remained lower in the positive group. The injection with oligomycin activated the glycolysis at its maximum level in the positive group, but did not affect the negative group.
The injection with the glucose analog at 2-DG returned the extracellular acidification rate to non-glycolytic levels. For the mitochondrial stress test, the injection of oligomycin hyperpolarized the mitochondrial membrane preventing more proton pumping through the ETC complexes and thus reducing the rate of mitochondrial respiration. The injection of FCCP pushes the oxygen consumption rate to the maximum as cells try to recover the mitochondrial membrane potential.
The injection with two other electron transport chain inhibitors causes mitochondrial respiration to completely stop and thus the oxygen consumption rate reverted to its minimum level. Please avoid the use of red cell lysis buffer for the mononuclear cell isolation. We recommend the Ficoll gradient separation to prevent clump formation and reduce the loss of LSK.
Using our optimized method, we can measure mitochondrial respiration and glycolysis of rare and fragile hematopoietic stem and progenitor cells and study of metabolic cues regulate normal and malignant hematopoiesis.
