We thank the UNIL Flow Cytometry Core Facility for their support especially Dr. Romain Bedel. This work was supported by the Kristian Gerhard Jebsen foundation grant to N.V and O.N.

All experiments described in the manuscript follow the guidelines of our institution and were carried out in accordance with Swiss law for animal experimentation (Authorization: VD3194) and for research involving human samples (Protocol: 235/14; CER-VD 2017-00490)
1. Hematopoietic Stem Cell Extraction
<ol>
	<li>Purchase wild type C57BL6/J mice and keep them in the animal house for at least a week to reduce transport-associated stress.</li>
	<li>On the day of the experiment, euthanize the mou...

<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=Durable+complete+responses+in+heavily+pretreated+patients+with+metastatic+melanoma+using+T+cell+transfer+immunotherapy.">Durable complete responses in heavily pretreated patients with metastatic melanoma using T cell transfer immunotherapy.</a> Clinical Cancer Research. 17 (13), 4550-4557 (2011).</li><li>Aranda, F., 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=Trial+Watch:+Adoptive+cell+transfer+for+anticancer+immunotherapy.">Trial Watch: Adoptive cell transfer for anticancer immunotherapy.</a> Oncoimmunology. 3, 28344 (2014).</li><li>Wherry, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+exhaustion.">T cell exhaustion.</a> Nature Immunology. 12 (6), 492-499 (2011).</li><li>Ho, P. C., 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=Phosphoenolpyruvate+Is+a+Metabolic+Checkpoint+of+Anti-tumor+T+Cell+Responses.">Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses.</a> Cell. 162 (6), 1217-1228 (2015).</li><li>Chang, C. H., 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=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>van der Waart, A. B., 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=Inhibition+of+Akt+signaling+promotes+the+generation+of+superior+tumor-reactive+T+cells+for+adoptive+immunotherapy.">Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy.</a> Blood. 124 (23), 3490-3500 (2014).</li><li>Crompton, J. G., 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=Akt+inhibition+enhances+expansion+of+potent+tumor-specific+lymphocytes+with+memory+cell+characteristics.">Akt inhibition enhances expansion of potent tumor-specific lymphocytes with memory cell characteristics.</a> Cancer Research. 75 (2), 296-305 (2015).</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>Abe, 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=Establishment+of+Conditional+Reporter+Mouse+Lines+at+ROSA26+Locus+For+Live+Cell+Imaging.">Establishment of Conditional Reporter Mouse Lines at ROSA26 Locus For Live Cell Imaging.</a> Genesis. 49 (7), 579-590 (2011).</li><li>Sukumar, 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=Mitochondrial+Membrane+Potential+Identifies+Cells+with+Enhanced+Stemness+for+Cellular+Therapy.">Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.</a> Cell Metabolism. 23 (1), 63-76 (2016).</li><li>Sukumar, 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=Inhibiting+glycolytic+metabolism+enhances+CD8++T+cell+memory+and+antitumor+function.">Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function.</a> Journal of Clinical Investigation. 123 (10), 4479-4488 (2013).</li></ol>

Some elements of this work have been submitted as application P1828EP00 to the European Patent Office.

A tight regulation of HSC function is important to maintain stable hematopoiesis during an organism&#39;s lifetime. Like various other cell types in the body, a key component that contributes to the regulation of HSC function is cellular metabolism. Previous studies from our lab9 and others2,3 have implicated the importance of mitochondria in maintaining a distinct metabolic state in HSCs. Due to the extremely low number of HSCs isolated f...

Hematopoietic stem cells (HSCs) are a small population of cells residing in the bone marrow ensuring blood production and homeostasis throughout an organism&#39;s lifetime. HSCs mediate this process by giving rise to progenitors that in turn produce terminally differentiated mature blood cell lineages via several rounds of cell division and well-orchestrated differentiation steps1. Importantly, HSCs produce their energy via anaerobic glycolysis. In contrast, more committed and active hematopoietic progenitors switch their metabolism toward mitochondrial metabolism2,3,4. This distinct metabolic state is believed to protect the HSCs from cellular damage inflicted by reactive oxygen species (ROS) produced by active mitochondria, thereby maintaining their long-term in vivo function5,6,7,8. Direct measurement of the HSC metabolic state is challenging and often low throughput due to their limited numbers. Here, we describe a flow cytometry-based assay for robust measurement of mitochondrial membrane potential (&#916;&#936;m) using tetramethylrhodamine methyl ester (TMRM) fluorescence, and mitochondrial mass using a green fluorescent mitochondrial stain (Mitotracker Green) in HSCs. We have previously demonstrated that low &#916;&#936;m is a bona-fide functional marker of highly purified HSCs9 and metabolic modulators capable of lowering &#916;&#936;m enhance HSCs function9,10. Here we propose use of our method on HSCs mitochondrial profiling as strategy to identify novel molecules capable of improving the HSCs&#39; long-term blood reconstitution potential.
As an example, we demonstrate that this assay reliably measures the lowering of HSC &#916;&#936;m upon exposure to vitamin B3 analog nicotinamide riboside (NR). Accordingly, in our recently published study we demonstrate that NR strongly ameliorates blood recovery posttransplant in both mouse and humanized mouse systems by directly improving hematopoietic stem and progenitor functions10. The capacity of such metabolic modulators is of great clinical value considering that a 25% death rate is linked to delay in blood and immune recovery in posttransplanted patients11,12.
Moreover, we provide evidence that this methodology can be applied for the characterization of the metabolic profile and function of human T cells. In recent years, the development of adoptive cell therapy (ACT) using autologous tumor infiltrating lymphocytes (TILs) has become the most effective approach for certain types of advanced cancer with extremely unfavorable prognosis (e.g., metastatic melanoma, where &#62;50% of patients respond to treatment and up to 24% of patients have complete regression)13. However, TILs harboring sufficient antitumor activity are difficult to generate14. The extensive proliferation and stimulation that TILs undergo during ex vivo expansion cause T cell exhaustion and senescence that dramatically impair T cell antitumor response15. Importantly, the TILs&#39; antitumoral capacity is tightly linked to their metabolism16,17 and approaches aimed to modulate metabolism through the inhibition of the PI3K/Akt pathway have produced encouraging results18,19. For this reason, we compare the &#916;&#936;m of T cells derived from peripheral blood mononuclear cells (PBMCs) and patient-derived TILs, and show that less differentiated PBMC-derived T cells have lower &#916;&#936;m and mitochondrial mass as compared to terminally differentiated TILs.
We envision that this assay can be used to identify novel metabolic modulators that improve HSC and T cell function via the modulation of &#916;&#936;m.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL FACS tubes</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>Sample preparation</td>
		</tr>
		<tr>
			<td>96-U bottom plate</td>
			<td>Corning</td>
			<td>3799</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>AutoMACS pro separator</td>
			<td>Miltenyi Biotec</td>
			<td></td>
			<td>Automatic Cell separation</td>
		</tr>
		<tr>
			<td>BD FACS AriaIII</td>
			<td>Becton and Dickinson</td>
			<td></td>
			<td>Cell sorting</td>
		</tr>
		<tr>
			<td>BD IMag mouse hematopoietic progenitor cell enrichment kit</td>
			<td>BD</td>
			<td>558451</td>
			<td>Lineage depletion</td>
		</tr>
		<tr>
			<td>BD LSRII</td>
			<td>Becton and Dickinson</td>
			<td></td>
			<td>FACS acquisition machine</td>
		</tr>
		<tr>
			<td>BD-DIVA</td>
			<td>Becton and Dickinson</td>
			<td></td>
			<td>Acquisition software</td>
		</tr>
		<tr>
			<td>CD150 PE</td>
			<td>Biolegend</td>
			<td>115904</td>
			<td>Antibody staining mix</td>
		</tr>
		<tr>
			<td>CD150 PE-Cy5</td>
			<td>Biolegend</td>
			<td>115912</td>
			<td>Antibody staining mix</td>
		</tr>
		<tr>
			<td>CD48 PB</td>
			<td>Biolegend</td>
			<td>103418</td>
			<td>Antibody staining mix</td>
		</tr>
		<tr>
			<td>Centrifuge- 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Ckit PeCy7</td>
			<td>Biolegend</td>
			<td>105814</td>
			<td>Antibody staining mix</td>
		</tr>
		<tr>
			<td>Flow jo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>FACS Analysis software</td>
		</tr>
		<tr>
			<td>GraphPad-Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td>Plotting data into graphs</td>
		</tr>
		<tr>
			<td>Mitotracker Green</td>
			<td>Invitrogen</td>
			<td>M7514</td>
			<td>Green-fluorescent mitocondrial stain to measure mitochondrial mass; working concentration = 100 nM; stock concentration = 1 mM</td>
		</tr>
		<tr>
			<td>Nicotinamide Riboside (NR)</td>
			<td>Custom synthesized in house</td>
			<td></td>
			<td>Metabolic modulator; working concentration = 500 &#181;M; stock concentration = 50 mM</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>CHUV</td>
			<td>1000324</td>
			<td>Buffer preparation; working concentration = 1x; stock concentration = 1x</td>
		</tr>
		<tr>
			<td>Pen-Strep (P/S)</td>
			<td>Life technologies</td>
			<td>15140122</td>
			<td>Ex vivo culture; working concentration = 1x; stock concentration = 1x</td>
		</tr>
		<tr>
			<td>RBC Lysis buffer</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td>Lysing Red blood cells; working concentration = 1x; stock concentration = 10x</td>
		</tr>
		<tr>
			<td>Recombinant Mouse Flt-3 Ligand (FLT3)</td>
			<td>RnD</td>
			<td>427-FL-005/CF</td>
			<td>Ex vivo culture; working concentration = 2 ng/mL; stock concentration = 10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Recombinant mouse stem cell factor (SCF)</td>
			<td>RnD</td>
			<td>455-MC-010/CF</td>
			<td>Ex vivo culture; working concentration = 100 ng/mL; stock concentration = 50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Sca1 APC</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-5981-82</td>
			<td>Antibody staining mix</td>
		</tr>
		<tr>
			<td>StemlineII Hematopoietic Stem Cell Expansion Medium</td>
			<td>SIGMA</td>
			<td>S0192</td>
			<td>Ex vivo culture</td>
		</tr>
		<tr>
			<td>Streptavidin Pac orange</td>
			<td>Life Technologies</td>
			<td>S32365</td>
			<td>Antibody staining mix</td>
		</tr>
		<tr>
			<td>Streptavidin Tx red</td>
			<td>Life Technologies</td>
			<td>S872</td>
			<td>Antibody staining mix</td>
		</tr>
		<tr>
			<td>TMRM</td>
			<td>Invitrogen</td>
			<td>T668</td>
			<td>Staining mitochondrial membrane potential; working concentration = 200 nM; stock concentration = 10 mM</td>
		</tr>
		<tr>
			<td>Ultra pure EDTA</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td>Buffer preparation; working concentration = 0.5 M; stock concentration = 1 mM</td>
		</tr>
	</tbody>
</table>

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.

In Figure 1 we show the gating strategy for the isolation of hematopoietic stem cells from the mouse bone marrow and the layout of the plate for their ex vivo culture. Figure 1A shows the identification of the lymphocyte fraction in the SSC-A/FSC-A plot. Doublets were removed in the singlet gate followed by identification of live cells by the absence of DAPI signal. The LKS population, defined by lineage- Sca1+cKit+, was id...

Watch this Scientific Journal Video about Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry at JoVE.com

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

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 ...

This protocol is a simple flow cytometry-based assay allowing measurement of mitochondrial membrane potential and mitochondrial mass in living cells. Standard metabolic assays fail when working with rare populations such as the hematopoietic stem cells. This method enables users mitochondrial membrane profiling when working with low number of cells and to determine novel metabolic modulators of cells such as the HSCs.Moreover, you can combine it with downstream functional assays such as bone marrow transplantation or colony-forming assays to determine the functionality of your cells post-culture. Our technique can be exploited for the identification of novel molecules capable to improve HSC function in the context of bone marrow transplantation and hematopoietic immune recovery after ablative therapies. As described in our protocol, our method can be used to evaluate metabolic fitness of immune T cells.It is very important that the cells have minimal exposure to light after staining. Moreover, the use of pump inhibitor verapamil has to be included in order to evaluate the contribution of pump efflux. However, users have to be aware that verapamil profoundly perturbs ionic equilibrium and cannot be used in order to evaluate the effect of a metabolic modulator on mitochondrial membrane potential.Visual demonstration of this method is extremely important because there are some critical steps that one needs to keep in mind when working with low number of cells, and these steps are very often missing in the regular papers that users read on PubMed. For FAC sorting, the LKS population, defined by lineage negative, Sca1 positive, cKit positive is identified. Hematopoietic stem cells from around five to 10%of the cells in the LKS population are identified by gating for CD150 positive, CD48 negative population, labeled as HSCs.After hematopoietic stem cell sorting, Centrifuge the tubes at 300 times g for five minutes at four degrees Celsius. Gently remove most of the supernatant without dislodging the pellet, and leave 50 to 80 microliters on top of the cell pellet. Resuspend the cell pellet in stem cell expansion medium to a final volume of 200 microliters.Prepare a sterile tissue culture treated 96 U-bottom well plate, and identify the wells where the cells will be cultured. Transfer 100 microliters of 2x basal medium previously prepared in these wells. In the NR marked well, add two microliters of a 100x nicotinamide riboside solution.Transfer 100 microliters of 2x basal medium into wells for staining control. Seed 100 microliters of prepared hematopoietic stem cells on top of the wells containing 2x basal medium. Seed 100 microliters of whole bone marrow cells on wells marked as staining controls.Add 200 microliters of autoclaved water in all surrounding wells to reduce evaporation from wells containing cells. Place the plate undisturbed in an incubator at 37 degrees Celsius in 5%carbon dioxide for 72 hours. Take out the plate to replenish nicotinamide riboside every 24 hours, and put it back into the incubator.At the end of the culture period, add two microliters of 100x TMRM solution and two microliters of 100x green fluorescent stain solution in each of the test wells. Add two microliters of 100x TMRM in the TMRM staining control well. Add two microliters of 100x green fluorescent stain in the MitoTracker staining control well.Cover the top of the plate with aluminum foil. and place the plate back in an incubator at 37 degrees Celsius in 5%carbon dioxide for 45 minutes. Remove the plate from the incubator, and centrifuge it at 300 times g for five minutes.Invert the plate to remove the supernatant, and add 200 microliters of standard FACS buffer to resuspend. Cover the plate with foil. Centrifuge the plate at 300 times g for five minutes.Repeat this washing step three times. Resuspend the cells in 200 microliters of FACS buffer, and transfer to FACS tubes. To run the samples on the flow cytometer, first load the single color controls into the machine, and record at least 5, 000 events in each one by one.In the software, press Acquire and then Record on the dashboard to acquire and record single color controls. Press Compensation Setup, and click Calculate Compensation to calculate the compensation. Once the compensation has been applied, acquire the hematopoietic stem cell sample, and record as many events as possible.Export the FACS files from the cytometer, and open the file on the analysis software. On FlowJo, use forward scatter and side scatter gating to identify the cell population. Identify singlets in the next gates, and then plot the DAPI negative fraction representing live cells.In the live cell gate, make a contour plot in the TMRM and green fluorescent stain channel to measure mitochondrial membrane potential and mass, respectively. Export the mean fluorescence intensity of these two channels in the live cell gate. Then, export the proportion of cells in the TMRM low gate in all samples.The nicotinamide riboside treatment showed a clear increase in the TMRM low population. It also significantly increased the proportion of cells in the TMRM low gate and showed a significant lowering of TMRM fluorescence intensity. Mitochondrial mass did not change upon nicotinamide riboside supplementation.Additionally, stem cell marker staining was combined with mitochondrial staining post-culture. With the gating strategy to identify hematopoietic stem cells, exposure to nicotinamide riboside showed a significant increase in the TMRM low population and a significant decrease in the TMRM fluorescence intensity in gated HSCs. The green fluorescent mitochondrial stain green signal remained unchanged in the two conditions.It is important to cover the staining plate with aluminum foil in order to minimize the exposure of light to stained samples. This method can be combined with downstream assays such as bone marrow transplantation and colony-forming assays to determine the functionality of your stem cells. So this technique allows a simple screening method for the identification of novel metabolic modulators of HSCs.

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11.8K visualizações.  University of Lausanne. Este protocolo é um simples ensaio baseado em citometria de fluxo que permite a medição do potencial da membrana mitocondrial e da massa mitocondrial em células vivas. Ensaios metabólicos padrão falham quando se trabalha com populações raras, como as células-tronco hematopoiéticas. Este método permite aos usuários traçar perfis de membrana mitocondrial ao trabalhar com baixo número de células e determinar novos moduladores metabólicos de células como os HSCs.Além diss...