The authors thank all members of the Ito laboratory, especially K Ito and H Sato, and the Einstein Stem Cell Institute for comments and the Einstein Flow Cytometry and Analytical Imaging core facilities (funded by National Cancer Institute grant P30 CA013330) for help carrying out the experiments. K.I. is supported by grants from the National Institutes of Health (R01DK98263, R01DK115577, and R01DK100689) and the New York State Department of Health as Core Director of Einstein Single-Cell Genomics/Epigenomics (C029154). K.I. Ito is a Research Scholar of the Leukemia and Lymphoma Society.

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
<ol>
	<li>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 &#176;C for at least one month in sterile condition. Before starting the following procedures, put an aliquot of this solution (50 mL) on ice.</li>
	<li>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.</li>
	<li>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).</li>
	<li>TMRM stock solution: Prepare TMRM (1 &#956;M) solution by dissolving 5 &#956;g of TMRM powder in 10 mL of ethanol. Store this solution at -20 &#176;C protected from the light.</li>
	<li>Verapamil stock solution: Prepare Verapamil (50 mM) solution by diluting 24 mg of Verapamil in 1 mL of ethanol. Store this solution at -20 &#176;C.</li>
	<li>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 &#176;C protected from the light.</li>
</ol>
2. Bone Marrow Isolation
<ol>
	<li>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.</li>
	<li>Using a pair of forceps and sharp scissors, make a small snip in the ventral skin of the mouse and stretch the skin.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Collect all cells in a 1.5 mL tube, centrifuge for 5 min at 180 x g then discard the supernatant (Figure 1C).</li>
	<li>Resuspend the pellet in 300 &#956;L 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.</li>
	<li>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 &#956;m nylon mesh incorporated to obtain mononuclear cells. After blocking, keep the sample on ice.</li>
</ol>
3. Immunostaining for Detection of HSC
<ol>
	<li>Prepare Lineage (Lin) cocktail. In 400 &#956;L of staining buffer, add 4 &#956;L 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.</li>
	<li>Prepare fluorophores conjugated-antibodies (Abs). In 400 &#956;L of staining buffer, add 4 &#956;L 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.</li>
	<li>Centrifuge the sample for 5 min at 180 x g, then discard the supernatant.</li>
	<li>Add 400 &#956;L of Lin cocktail solution to the cells pellet. Add 4 &#956;L of CD135-biotinilated Ab. Vortex quickly to mix and incubate for 30 min in ice.</li>
	<li>Wash the sample adding 3 mL of staining buffer, spin down for 5 min at 180 x g and discard the supernatant.</li>
	<li>Add 400 &#956;L of Abs solution. Vortex quickly to mix and incubate for 30 min on ice.</li>
	<li>Wash the samples with 3 mL of staining buffer, spin down for 5 min at 180 x g and discard the supernatant.</li>
</ol>
4. TMRM Staining
<ol>
	<li>Prepare TMRM staining solution. Add 2.2 &#956;L of TMRM stock solution and 1.1 &#956;L of Verapamil (2 nM and 50 &#956;M 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.</li>
	<li>Resuspend the bone marrow in 1 mL of TMRM staining solution, vortex quickly and incubate for 1 h at 37 &#176;C. 
	NOTE: TMRM staining must not be washed out. PE-dedicated compensation control is also resuspended in 100 &#956;L of TMRM staining solution, and subjected to incubation for 1 h at 37 &#176;C after quick vortex.</li>
	<li>Filter the sample using a cell-strainer cap (12 x 75 mm2, 5 mL of capacity) with a 35 &#956;m 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.</li>
	<li>Add 1 &#956;L of 4&#8217;,6-diamidino-2-phenylindole (DAPI) to exclude dead cells by flow cytometry.</li>
</ol>
5. Acquisition by Flow Cytometer
<ol>
	<li>Run bone marrow sample and acquire at least 1 x 106 events.</li>
	<li>Set up the gating strategy to identify the different hematopoietic populations (Figure 2).
	<ol>
		<li>Display live bone marrow mono-nuclear cells (BM-MNCs), DAPI&#8722; fraction, in plot for Pacific Blue to identify CD135&#8722;Lin&#8722; (Lin&#8722;) and Lin+ fractions.</li>
		<li>Plot Lin&#8722; fraction for APC/Cy7 (c-kit) versus PE/Cy7 (Sca-1) to identify multipotent progenitors (MPP) fraction, as c-kit+ and Sca-1+.</li>
		<li>Plot MPP fraction for APC (CD48) versus PerCP/Cy5.5 (CD150) to identify HSC fraction, as CD150+ and CD48&#8722;.</li>
		<li>Display HSC fraction for FITC (CD34) to divide CD34&#8722;-HSC and CD34+-HSC.</li>
	</ol>
	</li>
	<li>Acquire the TMRM intensity (PE channel) in each population.</li>
	<li>After acquisition, add to the sample 1 &#956;L of FCCP to obtain the final concentration of 1 mM and incubate at 37 &#176;C for 5 min, then acquire 1 x 106 events. 
	NOTE: FCCP is a mitochondrial uncoupler which is used to dissipate the &#916;&#936;m. 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).</li>
	<li>Analyze data normalizing the average intensity of PE of each population by the intensity of PE of all BM-MNCs.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+efflux+studies+suggest+that+hematopoietic+stem+cells+expressing+low+or+undetectable+levels+of+CD34+antigen+exist+in+multiple+species.">Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.</a> Nature Medicine. 3 (12), 1337-1345 (1997).</li><li>Spangrude, G. J., Johnson, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+and+activated+subsets+of+mouse+multipotent+hematopoietic+stem+cells.">Resting and activated subsets of mouse multipotent hematopoietic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (19), 7433-7437 (1990).</li><li>Scharenberg, C. W., Harkey, M. 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The authors have nothing to disclose.

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 &#916;&#936;m by TMRM staining. TMRM is a cell-permeant fluorescent dye which accumulates in active mitochondria due to &#916;&#936;m, and its respective levels remain in equilibrium between the extracellular, cytoplasmic and mitochondrial compartments10. This protocol can be adapted for various dyes, including tetramethylrhodamine, ethyl ester (TMRE) and JC-1. Appropriate staining conditions are critical to achieving accurate results. These include: protection from light exposure, proper incubation time, maintenance of constant temperature (37 &#176;C) and extracellular concentration. When TMRM dye levels have not yet reached perfect equilibrium, unexpected changes in staining intensity can be detected during data acquisition in the flow cytometry process. Therefore the recommended staining period for TMRM is 1 h rather than 30 min (as mentioned in one TMRM manufacturer&#8217;s protocol). It is also suggested that the sample should be acquired at least two times within 5-min in order to compare the stability of the dye.
Another critical step of the protocol is the setting up of an efficient color match among antibodies and dye. Hematopoietic populations can be detected by flow cytometry using well-established surface markers21.The gating strategy described here (Figure 2) is compatible with TMRM staining, and allows for the measurement of its intensity at different differentiation stages simultaneously. TMRM matches the PE channel, but its intensity is usually much weaker than that of antibodies conjugated with PE (data not shown). As this can affect color compensation, causing unexpected shifts in the spectra of some populations, the sample with TMRM staining should be used as a compensation control for the PE channel.
The major advantage of the current protocol is that it enables the removal of the effect of xenobiotic efflux pumps, whose efficient extrusion of TMRM dye modifies both its equilibrium across the plasma membrane and its mitochondrial accumulation. This is a critical step in the accurate assessment of HSC mitochondrial function by dye absorption. As HSCs express more active efflux pumps than committed cells19, Verapamil or similar drugs, such as Cyclosporine H, a Ca2+-independent multidrug resistance inhibitor22 (Figure 3B-C), should be used in the staining procedure for HSCs to more accurately assess levels of mitochondrial activity. Because this critical issue was pointed out only recently11,19 and is still under discussion23, the main aim of this report is to provide an easy and detailed protocol which will help to standardize the procedure for obtaining reproducible data and reduce the apparent discrepancies between the results of different research groups. The protocol here described shows in detail each step, including doses, timing and specific media. For instance, TMRM profiles in HSPCs can differ depending on their medium (Figure 3A). A detailed analysis of the mechanisms involved will require further exploration, but since serum-free expansion medium (SFEM) is a well-established method for HSC culture, SFEM rather than PBS is strongly recommended when performing TMRM staining for HSCs. Further, the accurate assessment of mitochondrial membrane potential demonstrated here through the inhibition of efflux pumps highlights possible future applications of Verapamil, as well as other dyes (e.g. MitoTracker, MitoSOX), in the investigation of HSCs.
Importantly, TMRM intensity as shown by flow cytometry may not precisely reflect mitochondrial volume. Flow cytometry measures total fluorescence intensity per cell, but the distribution of mitochondrial dyes to mitochondria depends on &#916;&#936;m. It is therefore difficult to discern whether the critical contribution to a TMRM reading derives from &#916;&#936;m or mitochondrial volume11. We have confirmed that MTO, one of the dyes most frequently used to measure mitochondrial mass, is affected by both &#916;&#936;m and efflux pump activity. We have also observed that Verapamil changes the intensity profile of MTO in HSPC populations, but have found no correlation between MTO intensity and mitochondrial volume11. Combined measurement of mitochondrial volume and &#916;&#936;m should be used to normalize data and eliminate the effects of membrane potential, and further exploration of mitochondrial content should be carried out using &#916;&#936;m-independent techniques.
Such techniques would include 3D volume analysis based on fluorescent markers (e.g., immunostaining of mitochondrial proteins), electron microscopy11, and quantitation of mitochondrial/nuclear DNA ratios. The use of mitochondrial dyes (i.e., TMRM) in combination with efflux system inhibitors will doubtless prove of great benefit in the elucidation of mitochondrial biology through flow cytometry.

Hematopoietic stem cells (HSCs) are self-renewing, multi-potent, and capable of giving rise to all the cells of the blood1,2. Cellular metabolism is a key regulator of HSC maintenance, along with transcriptional factors, intrinsic signals and the microenvironment3,4,5. The proper control of mitochondrial function and quality is therefore critical to HSC maintenance6,7.
Mitochondrial membrane potential (&#916;&#936;m) 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/FO ATP synthase. These are both required (depending on gene expression and substrate availability) for the oxygen-dependent phosphorylation of ADP to ATP8,9. Taking advantage of the electronegativity of the mitochondrial compartment, various potentiometric dyes have been developed to measure &#916;&#936;m. One of them is tetramethylrhodamine methyl ester perchlorate (TMRM), which has been extensively used to measure &#916;&#936;m by flow cytometry in a variety of cells10, including hematopoietic stem and progenitor cells11. &#160;
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 &#8220;side populations&#8221; by exploiting the differential extrusion of the dyes Hoechst Blue and Hoechst Red14,15. 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 &#916;&#936;m16,17,18.
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 &#916;&#936;m 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 &#916;&#936;m by potentiometric dyes. Here we describe a modified protocol for accurate &#916;&#936;m measurement by TMRM-based flow cytometry which corrects for xenobiotic transporter activity through the use of dedicated inhibitors.

<table><tbody><tr><td>ACK lysing buffer</td><td>Life Technologies</td><td>A1049201</td><td></td></tr><tr><td>B220-biotin</td><td>BD Bioscience</td><td>553086</td><td></td></tr><tr><td>CD3e-biotin</td><td>Life Technologies</td><td>13-0031-85</td><td></td></tr><tr><td>CD4-biotin</td><td>Fischer Scientific</td><td>BDB553782</td><td></td></tr><tr><td>CD8-biotin</td><td>Life Technologies</td><td>13-0081-85</td><td></td></tr><tr><td>CD11b-biotin</td><td>BD Bioscience</td><td>553309</td><td></td></tr><tr><td>CD19-biotin</td><td>BD Bioscience</td><td>553784</td><td></td></tr><tr><td>CD34-FITC</td><td>eBioscience</td><td>11-0341-85</td><td></td></tr><tr><td>CD48-APC</td><td>eBioscience</td><td>17-0481-82</td><td></td></tr><tr><td>CD135-biotin</td><td>eBioscience</td><td>13-1351-82</td><td></td></tr><tr><td>CD150-PerCP/Cy5.5&amp;nbsp;</td><td>Biolegend</td><td>115922</td><td></td></tr><tr><td>c-kit-APC/Cy7</td><td>Biolegend</td><td>105826</td><td></td></tr><tr><td>Cyclosporin H</td><td>Millipore Sigma</td><td>SML1575-1MG</td><td></td></tr><tr><td>DAPI solution (1 mg/mL)</td><td>Life Technologies</td><td>62248</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Denville</td><td>FB5001-H</td><td></td></tr><tr><td>FCCP</td><td>Millipore Sigma</td><td>C2920-10MG</td><td></td></tr><tr><td>Gr1-biotin</td><td>Biolegend</td><td>108404</td><td></td></tr><tr><td>IgM-biotin</td><td>Life Technologies</td><td>13-5790-85</td><td></td></tr><tr><td>Il7R&amp;alpha;-biotin</td><td>eBioscience</td><td>13-1271-85</td><td></td></tr><tr><td>Nk1.1-biotin</td><td>Fischer Scientific</td><td>BDB553163</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Life Technologies</td><td>10010023</td><td></td></tr><tr><td>Sca-1-PE/Cy7</td><td>eBioscience</td><td>25-5981-81</td><td></td></tr><tr><td>SCF murine</td><td>PEPROTECH</td><td>250-03-10UG</td><td></td></tr><tr><td>StemSpan SFEM medium</td><td>STEMCELL technologies</td><td>9605</td><td></td></tr><tr><td>Streptavidin-Pacific Blue</td><td>eBioscience</td><td>48-4317-82</td><td></td></tr><tr><td>Ter119-biotin</td><td>Fischer Scientific</td><td>BDB553672</td><td></td></tr><tr><td>TMRM</td><td>Millipore Sigma</td><td>T5428-25MG</td><td></td></tr><tr><td>TPO</td><td>PEPROTECH</td><td>315-14-10UG</td><td></td></tr><tr><td>Verapamil hydrochloride</td><td>Millipore Sigma</td><td>V4629-1G</td><td></td></tr></tbody></table>

improving the accuracy of flow cytometric assessment of mitochondrial membrane potential in hematopoietic stem and progenitor cells through the inhibition of efflux pumps

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 (&#916;&#936;m), 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 &#916;&#936;m 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 &#916;&#936;m-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.

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 the purification of HSCs (CD34&#8722; and CD34+), multipotent progenitor cells (MPPs), Lin&#8722; as well as Lin+ cells, respectively 21. The gating strategy for isolating these fractions is shown in Figure 2.
After the identification of populations of interest, TMRM intensity, which should appear as a bright signal, was assessed. TMRM staining in serum-free expansion medium (SFEM) is highly recommended, as TMRM profiles in HSPCs can undergo alteration when staining is performed in PBS +2% FBS (Figure 3A).
Figure 3C shows the average intensity of each population, which is normalized by the intensity of BM-MNCs. HSCs express high activity levels of xenobiotic efflux pumps capable of extruding TMRM dye20, and indeed, we found TMRM profiles in HSPCs were changed in the presence of Verapamil (Figure 3B,C). Similar results were obtained by other inhibitors such as Cyclosporin H (Figure 3C). Thus, the accurate amount of TMRM loaded in the mitochondria by &#916;&#936;m can be measured after inhibition of the efflux pumps by Verapamil or Cyclosporin H (Figure 3C).
Finally, FCCP can be used to verify the accuracy of TMRM staining. FCCP depolarizes mitochondria, resulting in a reduction in TMRM intensity (Figure 3D). This approach can also be used to determine the background intensity of the staining and/or as a negative control.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60057/60057fig1.jpg" /> 
Figure 1: Protocol flowchart. Graphical summary of the procedure to isolate and stain BM-MNCs to determine &#916;&#936;m. Critical steps are highlighted by picture inserts (A-D). Femurs and tibias from adult C57BL/6 mice were isolated and their ends are removed (A). Long bones as in A after flush out (B). Isolated BM-MNCs before (C) and after (D) ACK lysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60057/60057fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60057/60057fig2.jpg" /> 
Figure 2: Gating setup. Schematic representation of gating strategy to identify the different hematopoietic populations, including CD34&#8722;-HSC and CD34+-HSC, MPP, Lin&#8722; and Lin+ cells. The panels were modified from Bonora, M. et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60057/60057fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60057/60057fig3.jpg" /> 
Figure 3: Flow cytometry analysis of mitochondrial membrane potential. (A) Representative distribution of &#916;&#936;m in HSCs stained with TMRM in PBS+2%FBS (green) or in serum-free expansion medium (SFEM) (red). (B, C) Representative distribution of &#916;&#936;m in CD34&#8722;-HSC and Lin&#8722; cells (B) and quantification of &#916;&#936;m in CD34&#8722;-HSC, CD34+-HSC, MPP, Lin&#8722; and Lin+ cells (C) stained with TMRM in presence or absence of efflux pump inhibitors. TMRM intensity of each population was normalized by the TMRM intensity of own BM-MNCs (modified from Bonora, M. et al.11). (D) Representative histogram of TMRM intensity distribution in HSCs before (pink) and after (light blue) FCCP addition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60057/60057fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of 

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

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.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

improving-accuracy-flow-cytometric-assessment-mitochondrial-membrane

Nanyang Technological University

Research

JoVE Journal

Biology

7.7K Views.  Albert Einstein College of Medicine. Mitochondriale functie is van cruciaal belang voor hematopoietische stamcel zelfvernieuwing. Mitochondriale activiteit is een reflector van het binnenste mitochondriale membraanpotentieel, dat kan worden gemeten door TMRM, een cel fluorescerende, cel-permeant kationische kleurstof. Xenobiotische efflux pompen zijn zeer actief in HSC, en veroorzaken TMRM extrusie.Het belangrijkste voordeel van dit protocol, is de correctie van dit effect, met behulp van Verapamil, een remmer van de effl...

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

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Cancer Research

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Medicine

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

Immunology and Infection

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Neuroscience

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain&#39;s three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Author Spotlight: Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...

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

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