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 o...

<ol>
	<li>Weissman, I. L., Anderson, D. J., Gage, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+progenitor+cells:+origins,+phenotypes,+lineage+commitments,+and+transdifferentiations.">Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations.</a> Annual Review of Cell and Developmental Biology. 17, 387-403 (2001).</li><li>Morrison, S. J., Shah, N. M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+mechanisms+in+stem+cell+biology.">Regulatory mechanisms in stem cell biology.</a> Cell. 88 (3), 287-298 (1997).</li><li>Wilson, 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=Hematopoietic+stem+cells+reversibly+switch+from+dormancy+to+self-renewal+during+homeostasis+and+repair.">Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.</a> Cell. 135 (6), 1118-1129 (2008).</li><li>Morrison, S. J., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+marrow+niche+for+haematopoietic+stem+cells.">The bone marrow niche for haematopoietic stem cells.</a> Nature. 505 (7483), 327-334 (2014).</li><li>Frenette, P. S., Pinho, S., Lucas, D., Scheiermann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell:+keystone+of+the+hematopoietic+stem+cell+niche+and+a+stepping-stone+for+regenerative+medicine.">Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine.</a> Annual Review of Immunology. 31, 285-316 (2013).</li><li>Ito, K., Bonora, M., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+as+master+of+hematopoietic+stem+cell+fate.">Metabolism as master of hematopoietic stem cell fate.</a> International Journal of Hematology. 109 (1), 18-27 (2019).</li><li>Ito, K., 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=Self-renewal+of+a+purified+Tie2++hematopoietic+stem+cell+population+relies+on+mitochondrial+clearance.">Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.</a> Science. 354 (6316), 1156-1160 (2016).</li><li>Bonora, M., 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=ATP+synthesis+and+storage.">ATP synthesis and storage.</a> Purinergic Signal. 8 (3), 343-357 (2012).</li><li>Walker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+catalysis+in+ATP+synthase.">The regulation of catalysis in ATP synthase.</a> Current Opinion in Structural Biology. 4 (6), 912-918 (1994).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophysical Journal. 76, 469-477 (1999).</li><li>Bonora, M., Ito, K., Morganti, C., Pinton, P., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-potential+compensation+reveals+mitochondrial+volume+expansion+during+HSC+commitment.">Membrane-potential compensation reveals mitochondrial volume expansion during HSC commitment.</a> Experimental Hematology. 68, 30-37 (2018).</li><li>Goodell, M. 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. A., Torok-Storb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABCG2+transporter+is+an+efficient+Hoechst+33342+efflux+pump+and+is+preferentially+expressed+by+immature+human+hematopoietic+progenitors.">The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors.</a> Blood. 99 (2), 507-512 (2002).</li><li>Zhou, S., 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=The+ABC+transporter+Bcrp1/ABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype.">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nature Medicine. 7 (9), 1028-1034 (2001).</li><li>Simsek, T., 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=The+distinct+metabolic+profile+of+hematopoietic+stem+cells+reflects+their+location+in+a+hypoxic+niche.">The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche.</a> Cell Stem Cell. 7 (3), 380-390 (2010).</li><li>Vannini, N., 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=Specification+of+haematopoietic+stem+cell+fate+via+modulation+of+mitochondrial+activity.">Specification of haematopoietic stem cell fate via modulation of mitochondrial activity.</a> Nature Communications. 7, 13125 (2016).</li><li>Xiao, N., 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=Hematopoietic+stem+cells+lacking+Ott1+display+aspects+associated+with+aging+and+are+unable+to+maintain+quiescence+during+proliferative+stress.">Hematopoietic stem cells lacking Ott1 display aspects associated with aging and are unable to maintain quiescence during proliferative stress.</a> Blood. 119 (21), 4898-4907 (2012).</li><li>de Almeida, M. J., Luchsinger, L. L., Corrigan, D. J., Williams, L. J., Snoeck, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye-Independent+Methods+Reveal+Elevated+Mitochondrial+Mass+in+Hematopoietic+Stem+Cells.">Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells.</a> Cell Stem Cell. 21 (6), 725-729 (2017).</li><li>Hall, A. M., Rhodes, G. J., Sandoval, R. M., Corridon, P. R., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multiphoton+imaging+of+mitochondrial+structure+and+function+during+acute+kidney+injury.">In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury.</a> Kidney International. 83 (1), 72-83 (2013).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Nicolli, A., Basso, E., Petronilli, V., Wenger, R. M., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+cyclophilin+with+the+mitochondrial+inner+membrane+and+regulation+of+the+permeability+transition+pore,+and+cyclosporin+A-sensitive+channel.">Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel.</a> The Journal of Biological Chemistry. 271 (4), 2185-2192 (1996).</li><li>Vannini, N., 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=The+NAD-Booster+Nicotinamide+Riboside+Potently+Stimulates+Hematopoiesis+through+Increased+Mitochondrial+Clearance.">The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance.</a> Cell Stem Cell. 24 (3), 405-418 (2019).</li></ol>

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...

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&#160;</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 (1mg/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&#945;-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...

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 ...

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.

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JoVE Journal

Biology

7.6K Views.  Albert Einstein College of Medicine. 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...

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