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

Summary

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

Abstract

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (ΔΨm), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure ΔΨm across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when ΔΨm-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Introduction

Hematopoietic stem cells (HSCs) are self-renewing, multi-potent, and capable of giving rise to all the cells of the blood^1,2. Cellular metabolism is a key regulator of HSC maintenance, along with transcriptional factors, intrinsic signals and the microenvironment^3,4,5. The proper control of mitochondrial function and quality is therefore critical to HSC maintenance^6,7.

Mitochondrial membrane potential (ΔΨm) is a key parameter in the assessment of mitochondria as it directly reflects their functionality, which derives from the equilibrium of proton pumping activity in the electron transport chain and the proton flow through F₁/F_O ATP synthase. These are both required (depending on gene expression and substrate availability) for the oxygen-dependent phosphorylation of ADP to ATP^8,9. Taking advantage of the electronegativity of the mitochondrial compartment, various potentiometric dyes have been developed to measure ΔΨm. One of them is tetramethylrhodamine methyl ester perchlorate (TMRM), which has been extensively used to measure ΔΨm by flow cytometry in a variety of cells¹⁰, including hematopoietic stem and progenitor cells¹¹.  

Mitochondrial dyes must be used with some caution in HSCs, however, because the high activity of the xenobiotic efflux pumps of these cells can result in dye extrusion¹². Indeed, the extrusion of mitochondrial dyes such as Rhodamine 123 has allowed researchers to isolate HSCs¹³ or identify HSC “side populations” by exploiting the differential extrusion of the dyes Hoechst Blue and Hoechst Red^14,15. It has also been shown that Fumitremorgin C, a specific blocker of the ATP-binding cassette sub-family G member 2 (ABCG2) transporter, does not affect the staining pattern of MitoTracker in HSPCs¹⁶. After the publication of these results, multiple studies were performed using mitochondrial dyes in the absence of xenobiotic efflux pump inhibitors, leading to the widespread impression that HSCs have only a small number of mitochondria with low ΔΨm^16,17,18.

Recently, it was demonstrated, however, that Verapamil, a wide spectrum inhibitor of efflux pumps, significantly modifies the staining pattern of the mitochondrial dye MitoTracker Green¹⁹. This discrepancy is likely due to the fact that Fumitremorgin C is highly selective for Abcg2, while HSCs also express other transporters such as Abcb1a (which is only weakly sensitive to Fumitremorgin C)¹⁹. We have also reported that other mitochondrial dyes, such as TMRM, Nonyl acridine orange, and Mitotracker Orange (MTO) exhibit the same patterns as Mitotracker Green. More importantly, we have observed that the flow cytometric patterns of HSPCs reflect their ΔΨm in addition to mitochondrial mass¹¹.

The intake of TMRM dye strictly depends on the negative charge of mitochondria, but the resulting accumulation of dye is in constant balance between its intake and clearance by efflux pumps²⁰. The difference in xenobiotic efflux pump expression between HSCs and mature cell populations affects this balance and can lead to biased results. The use of dedicated inhibitors such as Verapamil should be considered in the analysis of ΔΨm by potentiometric dyes. Here we describe a modified protocol for accurate ΔΨm measurement by TMRM-based flow cytometry which corrects for xenobiotic transporter activity through the use of dedicated inhibitors.

Protocol

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.

Results

The protocol described above enables the easy isolation of BM-MNCs from a mouse model. Figure 1 summarizes the main steps of the protocol: bone isolation, flushing out of the bone marrow, red blood cell lysis, and antibody staining followed by TMRM staining to measure mitochondrial membrane potential in a specific hematopoietic population.

BM-MNCs contain several cell populations, including HSCs. The antibody cocktails used in this protocol are well-established in...

Discussion

Mitochondrial membrane potential measurement is a cornerstone of the analysis and assessment of mitochondria, which are critical to the metabolic state of the cell. Here, we describe a protocol for the analysis of ΔΨm by TMRM staining. TMRM is a cell-permeant fluorescent dye which accumulates in active mitochondria due to ΔΨm, and its respective levels remain in equilibrium between the extracellular, cytoplasmic and mitochondrial compartments¹⁰. This protocol can be adapted for...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACK lysing buffer	Life Technologies	A1049201
B220-biotin	BD Bioscience	553086
CD3e-biotin	Life Technologies	13-0031-85
CD4-biotin	Fischer Scientific	BDB553782
CD8-biotin	Life Technologies	13-0081-85
CD11b-biotin	BD Bioscience	553309
CD19-biotin	BD Bioscience	553784
CD34-FITC	eBioscience	11-0341-85
CD48-APC	eBioscience	17-0481-82
CD135-biotin	eBioscience	13-1351-82
CD150-PerCP/Cy5.5	Biolegend	115922
c-kit-APC/Cy7	Biolegend	105826
Cyclosporin H	Millipore Sigma	SML1575-1MG
DAPI solution (1 mg/mL)	Life Technologies	62248
Fetal Bovine Serum (FBS)	Denville	FB5001-H
FCCP	Millipore Sigma	C2920-10MG
Gr1-biotin	Biolegend	108404
IgM-biotin	Life Technologies	13-5790-85
Il7Rα-biotin	eBioscience	13-1271-85
Nk1.1-biotin	Fischer Scientific	BDB553163
Phosphate buffered saline (PBS)	Life Technologies	10010023
Sca-1-PE/Cy7	eBioscience	25-5981-81
SCF murine	PEPROTECH	250-03-10UG
StemSpan SFEM medium	STEMCELL technologies	9605
Streptavidin-Pacific Blue	eBioscience	48-4317-82
Ter119-biotin	Fischer Scientific	BDB553672
TMRM	Millipore Sigma	T5428-25MG
TPO	PEPROTECH	315-14-10UG
Verapamil hydrochloride	Millipore Sigma	V4629-1G