Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

Mitochondrial function is critical for hematopoietic stem cell self renewal. Mitochondrial activity is a reflector of the inner mitochondrial membrane potential, which can be measured by TMRM, a cell fluorescent, cell-permeant cationic dye. Xenobiotic efflux pumps are highly active in HSC, and cause TMRM extrusion.

The main advantage of this protocol, is the correction of this effect, utilizing Verapamil, an inhibitor of the efflux pumps. Begin by isolating bone marrow from mouse femurs. Use a pair of forceps and sharp scissors to cut the two posterior paws.

Then make a small snip in the ventral skin of the mouse, and stretch it. Extract the femur and tibia, while taking care not to dislodge the heads of the femur, and place the bones in a six-well plate, filled with 1.5 milliliters of staining buffer. Remove the muscles from the bones, and cut the ends of the bones, allowing bone marrow to exit.

Place the cleaned bones in new wells, with 1.5 milliliters of staining buffer. Then, flush out the bone marrow with a three milliliter syringe and a 25 gauge needle, until the bone becomes white. Collect all the cells in a 1.5 milliliter tube, and centrifuge them for five minutes at 180 times G, then discard the supernatant.

Carefully resuspend the pellet in 300 microliters of ice cold ACK lysing buffer, and put the cells on ice for one minute. Then immediately deactivate lysis by adding one milliliter of staining buffer. Centrifuge the cells for five minutes at 180 times G.Resuspend the cell pellet, which should appear white, in one milliliter of staining buffer.

Filter the cells using a cell strainer cap, with a 35 micrometer nylon mesh, to obtain mononuclear cells. Start by preparing the lineage cocktail, and fluorophore conjugated antibodies, according to manuscript directions. Keep the antibodies on ice, and protected from light.

Then, centrifuge the sample for five minutes at 180 times G, and discard the supernatant. Add 400 microliters of the lineage cocktail, and four microliters of CD135 biotinylated antibodies to the cell pellet, and vortex the mixture. Then incubate it on ice for 30 minutes.

After the incubation, wash the sample by adding three milliliters of staining buffer, spinning it down for five minutes at 180 times G, and discarding the supernatant. Then add 400 microliters of antibody solution, and vortex the mixture. Incubate it on ice for another 30 minutes, and repeat the wash with the staining buffer.

To prepare the TMRM staining solution, add 2.2 microliters of TMRM stock solution, and 1.1 microliters of Verapamil, to 1.1 milliliters of serum-free hematopoietic culture medium, with thrombopoietin and stem cell factor. Resuspend the bone marrow in one milliliter of the TMRM staining solution, vortex quickly, and incubate for one hour at 37 degrees Celsius. Filter the sample using a cell strainer cap, with a 35 micrometer nylon mesh, to avoid clogging the flow cytometer.

Then add one microliter of DAPI and proceed with flow cytometry. Run the bone marrow sample, and acquire at least one million events. Then display the live bone marrow mononuclear cells in a plot for pacific blue, to identify CD135 Lin-negative and Lin-positive fractions.

Plot the Lin-negative fraction for APC/Cy7 versus PE/Cy7 to identify the multipotent progenitor or MPP fraction. Then plot the MPP fraction for APC versus PerCP/Cy5.5, to identify the hematopoietic stem cell or HSC fraction. Display the HSC fraction for FITC, or CD34, to divide it into CD34 negative HSC and CD34 positive HSC fractions.

Finally acquire the TMRM intensity in each population. To ensure that TMRM staining worked correctly, add one microliter of FCCP to the sample, for a final concentration of one millimolar, and incubate it at 37 degrees Celsius for five minutes. Then acquire one million events.

After FCCP addition, TMRM intensity should be drastically increased. This protocol has been successfully used to quantify mitochondrial membrane potential in various cell populations, with TMRM dye. It has been demonstrated that the presence of PBS and FBS in the culture medium affects TMRM signal intensity.

So it is important to perform TMRM staining in serum-free expansion medium. Hematopoietic stem cells express high activity levels of xenobiotic efflux pumps that extrude TMRM dye. So TMRM profiles also vary in the presence of Verapamil or Cyclosporin H, which block the pumps.

To verify the accuracy of TMRM staining, FCCP can be to depolarize the mitochondria, and reduce TMRM intensity. This protocol can be easily adapted for studying HSC with a different mitochondrial membrane potential dye, including TMRE or JC-1. As well as other mitochondrial dyes such as MitoTracker or MitoSOX.

The critical issues inherited by the different activity of efflux pumps among bone marrow population was pointed out only recently. So this protocol is aimed to reduce the discrepancy between previous findings.