All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Albert Einstein College of Medicine.

1. Preparation of Solutions

1. Staining Buffer (phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS)): Add 10 mL of FBS in 500 mL of a sterile PBS solution.
   NOTE: This solution can be stored at 4 °C for at least one month in sterile condition. Before starting the following procedures, put an aliquot of this solution (50 mL) on ice.
2. ACK (ammonium-chloride-potassium) lysing buffer: Place an aliquot of ACK lysing buffer (1 mL) on ice before starting the procedure.
   NOTE: To prepare the same non-commercial buffer, dissolve 8.02 g of NH₄Cl, 1 g of KHCO₃ and 37.2 mg of Na₂EDTA in 1 L of H₂O. Adjust the pH to 7.2-7.4. Store for up to 6 months at room temperature.
3. Culture medium: Add 50 ng/mL stem cell factor (SCF) and 50 ng/mL thrombopoietin (TPO) to serum-free medium for culture and expansion of hematopoietic cells (see Table of Material for commercial medium recommended).
4. TMRM stock solution: Prepare TMRM (1 μM) solution by dissolving 5 μg of TMRM powder in 10 mL of ethanol. Store this solution at -20 °C protected from the light.
5. Verapamil stock solution: Prepare Verapamil (50 mM) solution by diluting 24 mg of Verapamil in 1 mL of ethanol. Store this solution at -20 °C.
6. FCCP stock solution: Prepare carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) (1 M) solution by dissolving 254 mg in 1 mL of ethanol. Store this solution at -20 °C protected from the light.

2. Bone Marrow Isolation

1. Euthanize the mouse by CO₂ inhalation following institutional guidelines and spray the mice with 70% ethanol.
   NOTE: This step prevents contamination of the cells of interest without compromising experimental results.
2. Using a pair of forceps and sharp scissors, make a small snip in the ventral skin of the mouse and stretch the skin.
3. Extract the femur and tibia while taking care not to dislodge the heads of the femur as they contain a large amount of bone marrow cells. Place the removed bones in a 6-well plate filled with 1.5 mL of staining buffer.
   NOTE: This procedure does not require sterile area.
4. Remove the muscles from the bones and cut the ends of the bones allowing the exit of the bone marrow (Figure 1A). Place the cleaned bones in new wells with 1.5 mL of staining buffer.
   NOTE: Muscles and other tissues must be carefully removed to avoid syringe clogging in the next step.
5. Flush out the bone marrow using a 3 mL syringe with a 25 G needle.
   NOTE: Continue to flush the bone marrow until the bone becomes white (Figure 1B).
6. Collect all cells in a 1.5 mL tube, centrifuge for 5 min at 180 x g then discard the supernatant (Figure 1C).
7. Resuspend the pellet in 300 μL of ice cold ACK lysing buffer very carefully, put on ice for 1 min and immediately inactive lysis by adding 1 mL of staining buffer. Centrifuge for 5 min at 180 x g.
   NOTE: The cells pellet appears white (Figure 1D). The total number of cells isolated is about 2-4 x 10⁷.
8. Resuspend the pellets in 1 mL of staining buffer and filter using a cell-strainer cap (12 x 75 mm², 5 mL of capacity) with a 35 μm nylon mesh incorporated to obtain mononuclear cells. After blocking, keep the sample on ice.

3. Immunostaining for Detection of HSC

1. Prepare Lineage (Lin) cocktail. In 400 μL of staining buffer, add 4 μL of the following biotinilated antibodies against CD3e, CD4, CD8, B220, CD11b, Gr1, Ter119, CD19, Nk1.1, IgM and Il7Ra to obtain a final dilution 1:100.
2. Prepare fluorophores conjugated-antibodies (Abs). In 400 μL of staining buffer, add 4 μL of the following Abs to obtain a final dilution 1:100. Streptavidin-Pacific Blue, Sca1-PE/Cy7, c-kit-APC/Cy7, CD48-APC, CD150-PerCP/Cy5.5 and CD34-FITC. Keep in ice, protected from light.
3. Centrifuge the sample for 5 min at 180 x g, then discard the supernatant.
4. Add 400 μL of Lin cocktail solution to the cells pellet. Add 4 μL of CD135-biotinilated Ab. Vortex quickly to mix and incubate for 30 min in ice.
5. Wash the sample adding 3 mL of staining buffer, spin down for 5 min at 180 x g and discard the supernatant.
6. Add 400 μL of Abs solution. Vortex quickly to mix and incubate for 30 min on ice.
7. Wash the samples with 3 mL of staining buffer, spin down for 5 min at 180 x g and discard the supernatant.

4. TMRM Staining

1. Prepare TMRM staining solution. Add 2.2 μL of TMRM stock solution and 1.1 μL of Verapamil (2 nM and 50 μM as final concentration, respectively) in 1.1 mL of serum-free medium for culture and expansion of hematopoietic cells (see Table of Material for commercial medium recommended) with TPO and SCF.
   NOTE: This is the most important step of the protocol. Verapamil addition is necessary to block the efflux pumps which are highly expressed in the HSCs and can extrude TMRM.
2. Resuspend the bone marrow in 1 mL of TMRM staining solution, vortex quickly and incubate for 1 h at 37 °C.
   NOTE: TMRM staining must not be washed out. PE-dedicated compensation control is also resuspended in 100 μL of TMRM staining solution, and subjected to incubation for 1 h at 37 °C after quick vortex.
3. Filter the sample using a cell-strainer cap (12 x 75 mm², 5 mL of capacity) with a 35 μm nylon mesh incorporated to avoid clogging of the flow cytometer.
   NOTE: TMRM staining increases the possibility of clog formation in the sample. Filtration of the sample just before flow assays is recommended.
4. Add 1 μL of 4’,6-diamidino-2-phenylindole (DAPI) to exclude dead cells by flow cytometry.

5. Acquisition by Flow Cytometer

1. Run bone marrow sample and acquire at least 1 x 10⁶ events.
2. Set up the gating strategy to identify the different hematopoietic populations (Figure 2).
   1. Display live bone marrow mono-nuclear cells (BM-MNCs), DAPI⁻ fraction, in plot for Pacific Blue to identify CD135⁻Lin⁻ (Lin⁻) and Lin⁺ fractions.
   2. Plot Lin⁻ fraction for APC/Cy7 (c-kit) versus PE/Cy7 (Sca-1) to identify multipotent progenitors (MPP) fraction, as c-kit⁺ and Sca-1⁺.
   3. Plot MPP fraction for APC (CD48) versus PerCP/Cy5.5 (CD150) to identify HSC fraction, as CD150⁺ and CD48⁻.
   4. Display HSC fraction for FITC (CD34) to divide CD34⁻-HSC and CD34⁺-HSC.
3. Acquire the TMRM intensity (PE channel) in each population.
4. After acquisition, add to the sample 1 μL of FCCP to obtain the final concentration of 1 mM and incubate at 37 °C for 5 min, then acquire 1 x 10⁶ events.
   NOTE: FCCP is a mitochondrial uncoupler which is used to dissipate the ΔΨm. It is used as experimental control to confirm that TMRM staining works correctly. After the administration of FCCP, the TMRM intensity should drastically be decreased (Figure 3A).
5. Analyze data normalizing the average intensity of PE of each population by the intensity of PE of all BM-MNCs.