This work is in part supported by the funding support from the National Institutes of Health (HL131645, CA016058), the St. Baldrick&#8217;s Foundation, and the Pelotonia Foundation.

This protocol was approved by Nationwide Children&#8217;s Hospital Animal Care and Use Committee (IACUC).
NOTE: The protocol is described in chronological order that spans over the period of two days. Use fresh reagents as described in the protocol below.
1. Preparation of Reagents on the Day Prior to the Assay
<ol>
	<li>Hydrate the sensor cartridge.
	<ol>
		<li>Incubate 5 mL of the calibrant (Table of Materials) in a non-CO2 37 &#176;C incubator overnight.</li>
		<li>Open the flux assay kit (Table of Materials) and separate the sensor cartridge (green; top) from the utility plate (transparent; bottom). Next, place the sensor cartridge upside down and adjacent to the utility plate.</li>
		<li>Using sterile water, fill the wells of the utility plate (200 &#181;L) and the chambers around the wells (400 &#181;L).</li>
		<li>Submerge the sensor cartridge into the utility plate making sure that the sensors are completely covered by water. Tap 3x to avoid the formation of bubbles.</li>
		<li>Place the cartridge submerged in the utility plate in a non-CO2 37 &#176;C incubator overnight. To prevent evaporation of the water, make sure that the incubator is properly humidified. Place an open beaker containing H2O as an extra precaution next to the cartridge-utility plate, particularly if using a regular oven.</li>
	</ol>
	</li>
	<li>Prepare the assay plate.
	<ol>
		<li>Sterilize the surface of biosafety cabinet class 2 using 70% ethanol. Open the assay plate under the hood.</li>
		<li>Add 40 &#181;L of commercially available 0.01% (w/v) poly-L-lysine (PLL) solution to each well of the assay plate under the hood.</li>
		<li>Cover the assay plate with the lid provided in the kit. Incubate the closed assay plate at room temperature (RT) under the hood for 1 h. 
		NOTE: The goal of the incubation is to let PLL coat the surface of the assay plate to facilitate adhesion of suspension cells to the surface of the assay plate.</li>
		<li>After 1 h incubation of the assay plate, remove the excess solution with a sterile vacuum-based aspirator and air dry the well under the hood. 
		NOTE: It takes ~30&#8722;60 min to air-dry the wells of the assay plate following the excessive PLL removal using the aspirator.</li>
	</ol>
	</li>
</ol>
2. Day of the Assay
<ol>
	<li>Prepare the cartridge.
	<ol>
		<li>Lift the sensor cartridge. Place it upside down in the tissue culture hood and discard the water from the utility plate.</li>
		<li>Fill the utility plate wells with 200 &#956;L of the pre-warmed calibrant.</li>
		<li>Fill the chambers around the wells with 400 &#956;L of the calibrant.</li>
		<li>Submerge the sensor cartridge into the utility plate, making sure that the sensors are completely covered by the calibrant. Tap 3x to avoid the formation of bubbles.</li>
		<li>Equilibrate the sensor cartridge submerged in the utility plate in a non-CO2 37 &#176;C incubator for 45&#8722;60 min.</li>
	</ol>
	</li>
	<li>Harvest murine bone marrow-derived LSK HSPCs.
	<ol>
		<li>To accommodate sufficient biological replicates, plan to use HSPCs derived from one mouse per well of the assay plate. 
		NOTE: This bone marrow-harvesting method provides ~50,000&#8722;80,000 LSK HSPCs from each mouse. This protocol to measure extracellular flux is optimized for ~70,000 LSK HSPCs per well of a 96 well-plate.</li>
		<li>Euthanize mice using CO2 overdose and cervical dislocation, following local IACUC approved methods.</li>
		<li>Sterilize the surface of dissecting tools and the bench using 70% ethanol.</li>
		<li>For each mouse, pre-fill one Petri dish with 1x phosphate-buffered saline (PBS) containing 2% heat inactivated fetal bovine serum (FBS) at RT. 
		NOTE: Do not use pre-chilled PBS as it will create clumps in the following steps.</li>
		<li>Spray 70% ethanol (v/v) on the entire euthanized mouse. Isolate all bones, including upper and lower limbs, hip bones, sternum, rib cage, and spine, from the mouse17,18. Pull out white matter from the spinal cord of the mouse as it can contaminate LSK cells. Place all bones in 1x PBS (+ 2% FBS), as they are being collected, in a Petri dish until further use.</li>
		<li>Invert a 50 mL conical tube (new each time) and use it to triturate bones submerged in 1x PBS (+ 2% FBS) in the Petri dish.</li>
		<li>Using a 10 mL serological pipette, pipette up and down (~10x) to uniformly flush out cells from bones, after crushing bones.</li>
		<li>Place a 40 &#181;m cell strainer on a 50 mL conical tube. Pre-wet the surface of the strainer by passing 1 mL of 1x PBS (+ 2% FBS).</li>
		<li>Harvest bone marrow-derived cell suspension from step 2.2.7 and pass it through the pre-wet surface of the cell strainer to remove bone debris.</li>
		<li>Repeat steps 2.2.6 through 2.2.9 until all bone materials turn white as a marker that most of bone marrow cells are collected in the 50 mL conical tube. 
		NOTE: It often takes two 50 mL conical tubes per mouse to collect all bone marrow-derived cells.</li>
		<li>Centrifuge 50 mL conical tubes containing bone marrow-derived cells for 5 min at 500 x g and RT.</li>
		<li>Remove the supernatant. Resuspend bone marrow-derived cells (combine contents of both 50 mL tubes) in 5 mL of 1x PBS (+ 2% FBS) as a final volume and keep cells at RT.</li>
	</ol>
	</li>
	<li>Harvest mononucleated murine bone marrow cells.
	<ol>
		<li>Add 5 mL of density gradient medium (i.e., Ficoll) to a 15 mL conical tube. Then slowly add 5 mL of the bone marrow cell suspension. Make sure that cells remain as a layer above the density gradient medium.</li>
		<li>Centrifuge for 30 min at 500 x g and RT. Do not use a brake in the centrifuge. Make sure the centrifuge is at the lowest possible acceleration (e.g., 1 acceleration and 0 deceleration).</li>
		<li>Harvest the middle interface of mononucleated cells (white color) following centrifugation into a fresh 15 mL conical tube.</li>
		<li>Wash cells, harvested from density gradient medium, with 5 mL of 1x PBS (+ 2% FBS). Centrifuge for 5 min at 500 x g and 4 &#176;C. Remove the supernatant.</li>
		<li>Repeat step 2.3.4.</li>
		<li>Resuspend cell contents of the tube in 300 &#181;L of 1x PBS (+ 2% FBS). Aliquot 10 &#181;L of cell suspension for unstained or single-color control in a FACS tube.</li>
	</ol>
	</li>
	<li>Harvest LSK HSPCs from mononucleated murine bone marrow cells.
	<ol>
		<li>Make a cocktail of biotin-antibodies by mixing 3 &#181;L per sample of the following antibodies: Gr1, Cd8a, Cd5, B220, Ter119. Add 15 &#181;L of the biotin-antibody cocktail to 300 &#181;L of mononucleated bone marrow cells. 
		NOTE: Each antibody is used at 1:100 dilution.</li>
		<li>Incubate cells with the biotin-antibody cocktail for 30 min at 4 &#176;C with agitation to avoid cells clumping in the bottom of the tube.</li>
		<li>Add 10 mL of pre-chilled 1x PBS (+ 2% FBS) to cells mixed with the biotin-antibody cocktail.</li>
		<li>Centrifuge the tube for 5 min at 500 x g and 4 &#176;C. Discard the supernatant and resuspend the cell pellet in 400 &#181;L of 1x PBS (+ 2% FBS). Aliquot 10 &#181;L for streptavidin-single color control.</li>
		<li>Briefly vortex anti-biotin microbeads (Table of Materials) before use. Add 80 &#181;L of microbeads to each cell sample (of 400 &#181;L). Mix well and incubate for additional 20 min at 4 &#176;C, with agitation.</li>
		<li>Add 10 mL of pre-chilled 1x PBS (+ 2% FBS) to cells. Centrifuge the tube for 5 min at 500 x g and 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the cell pellet in 1 mL of 1x PBS (+ 2% FBS). Store at 4 &#176;C while setting up magnetic separation unit.</li>
		<li>Place a column (Table of Materials) in the magnetic field of the magnetic assisted cell sorting (MACS) separator at 4 &#176;C. Prepare the column for magnetic separation by rinsing it with 3 mL of 1x PBS (+ 2% FBS) under the gravity flow at 4 &#176;C.</li>
		<li>Add the cell suspension from step 2.4.7 to the pre-wet column at 4 &#176;C. Allow the cells to pass through the column at 4 &#176;C and collect effluent in a 15 mL conical tube. 
		NOTE: The fraction with unlabeled cells in such effluent represents the enriched lineage negative cells.</li>
		<li>Wash column with 3 mL of 1x PBS (+ 2% FBS) at 4 &#176;C. Repeat 3x. Collect the flow-through and keep it at 4 &#176;C. Count the eluted viable cells by trypan blue exclusion using a hemocytometer.</li>
		<li>Centrifuge the 15 mL conical tube containing the flow-through for 5 min at 500 x g and 4 &#176;C. Discard the supernatant. Resuspend cells in 0.5 mL of 1x PBS (+ 2% FBS) and transfer the contents to a FACS tube.</li>
		<li>Add 24 &#181;L of the LSK antibody cocktail to each 107 cells. The antibody cocktail contains equal concentration of 450-streptavidin antibody, PE-CY7-Sca1 antibody, and APC-c-Kit antibody.</li>
		<li>Incubate for 1 h at 4 &#176;C with agitation under dark (covered with tin foil).</li>
		<li>Add 3 mL of 1x PBS (+ 2% FBS) to the FACS tube. Centrifuge for 5 min at 500 x g and 4 &#176;C. Discard the supernatant.</li>
		<li>Resuspend antibody-labelled cells in 1 mL of 1x PBS (+ 2% FBS). Add 1 &#181;L of 1 mg/mL propidium iodide to cell suspension just before sorting.</li>
		<li>Filter contents of the FACS tube using a 40 &#181;m strainer right before sorting LSK cells.</li>
		<li>Collect LSK cells, via FACS sorting, into 1.5 mL tube containing 0.5 mL of complete media supplemented with 2 mM glutamine, 3 mg/mL glucose, 1 mM pyruvate, 1x thrombopoietin (TPO), 1x stem cell factor (SCF), 0.5x penicillin/streptomycin (P/S), pH 7.4 (Table 1).</li>
	</ol>
	</li>
</ol>
3. Mitochondrial Respiration and Glycolysis Assays of LSK HSPCs
<ol>
	<li>LSK seeding in the assay plate
	<ol>
		<li>Centrifuge LSK cells from step 2.4.17 for 5 min at 500 x g and RT. Discard the supernatant.</li>
		<li>Resuspend cells in complete media to a final concentration of at least 70,000 cells/40 &#181;L.</li>
		<li>Seed contents of 40 &#181;L media (containing 70,000 cells) in the PLL-coated 8-well plate from step 1.2.4. Leave all corner wells empty.</li>
		<li>Centrifuge for 1 min at 450 x g and RT. Do not apply the brake. Make sure that cells are attached to the well bottom using the inverted microscope.</li>
		<li>Add 135 &#181;L of complete media to the cells in each well for a final volume of 175 &#181;L. Add 175 &#181;L of complete media to the 2 corners of the plate as blanks.</li>
		<li>Incubate the cells in the non-CO2 incubator for 2 h at 37 &#176;C.</li>
		<li>Set up a program to add drugs to each well of the well plate in the analyzer using a metric described in Table 2 (mitochondrial stress test) and Table 3 (glycolysis stress test). 
		NOTE: While cells are incubating, turn on the instrument and make sure it is at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Mitochondrial stress test
	<ol>
		<li>Prepare 45 &#181;M stock solutions of oligomycin, 50 &#181;M carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP) and 25 &#181;M stock solutions of rotenone/antimycin A. To prepare 45 &#181;M oligomycin stock solution, dissolve the contents of the commercially available pouch in 280 &#181;L of the complete media. To prepare 50 &#181;M FCCP stock solution, dissolve the contents of the commercially available pouch in 288 &#181;L of the complete media. To prepare 25 &#181;M rotenone/antimycin A stock solution, dissolve the contents of the commercially available pouch in 216 &#181;L of the complete media.</li>
		<li>Take the sensor cartridge in the utility plate out from the incubator and load its ports (A, B, and C) such that each well would have final concentration of 2 &#181;M oligomycin, 1.5 &#181;M FCCP, and 0.5 &#181;M of rotenone/antimycin A as needed, following dilution metrics described in Table 4 (for oxygen consumption rate).</li>
		<li>Remove the lid from the sensor cartridge assembled in the utility plate and place it on the instrument tray. Start the calibration that will take 20 min.</li>
		<li>After the calibration, remove the utility plate and substitute it with assay plate containing LSK cells, which are now adhered to the bottom of the well.</li>
		<li>Press Continue to start the program described in Table 2. After the completion of the program, retrieve the data and analyze them using the Wave Desktop software. 
		NOTE: The data generated from the extracellular flux assays can be plotted using the dot plot from the Wave Desktop software or exported to web-based statistics program.</li>
	</ol>
	</li>
	<li>Glycolysis stress test
	<ol>
		<li>Reconstitute glucose in 300 &#181;L (100 mM), oligomycin in 288 &#181;L (50 &#181;M), 2-D glucose (2-DG) in 300 &#181;L (500 mM) of complete media from the commercially available pouch.</li>
		<li>Gently pipette up and down (~10x) to solubilize the compounds. Vortex the 2-DG for approximately 1 min to ensure proper dissolution into media.</li>
		<li>Remove the sensor cartridge out from the incubator. Load ports A through C following dilution metrics described in the Table 5 to obtain a final concentration of 10 mM glucose, 2 &#181;M oligomycin, and 50 mM 2-DG.</li>
		<li>Repeat steps 3.2.3 and 3.2.4.</li>
		<li>Press Continue to start the program described in Table 3. After the completion of the program, retrieve the data and analyze them using the Wave Desktop software.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, we demonstrate the isolation of a maximum amount of pure and viable murine LSK HSPCs population as well as the measurement of their glycolysis and mitochondrial respiration with an extracellular flux analyzer. Specifically, the protocol overcomes the following technical issues for the use of LSK HSPCs : i) the low frequency of LSK HSPCs in murine bone marrow14, ii) low basal metabolic activity of LSK HSPCs26, iii) the fragility of LSK HSPCs27, and iv) the non-adherence of LSK HSPCs to culture vessels15. In addition, we have optimized the drug concentrations and media composition for the optimum performance of the extracellular flux assays.
Bone marrow-derived HSPCs reside in the hypoxic niche and they display a glycolytic phenotype, which is crucial for the maintenance of their stemness28. Conversely, respiration is essential for HSPC differentiation6. As metabolic dysfunction of HSPCs leads to blood diseases; here, we have described the protocol and video-based tutorials to measure the hallmarks of metabolic functions, such as the OCR and the ECAR, for murine bone marrow-derived LSK HSPCs.
Contrary to manufacturer recommendations for adherent cells, we found that the glycolysis stress test results on LSK HSPCs are optimum when cells are cultured in glucose/pyruvate+ media compared to glucose/pyruvate- media. We realized that the lack of glucose in media for LSK HSPCs results in cell death during the ~2 h equilibration period in a non-CO2 incubator. As the glucose injection did not change the basal level of ECAR for LSK HSPCs cultured in glucose/pyruvate+ media, our modification of the protocol not only preserves the essence of the glycolytic stress test, but it also allows us to distinguish between the basal glycolysis (before any injections), glycolytic capacity (after oligomycin injection) and non-glycolytic acidification (after injection with 2-DG) of LSK HSPCs.
Our mitochondrial stress test of LSK HSPCs showed that the ATP produced by oxidative phosphorylation in LSK HSPCs is minimal as seen with the small reduction in the OCR after the oligomycin injection. Conversely, the elevation in the OCR upon the FCCP injection affirms that LSK HSPCs are able to respond to higher energy demand.
Together, the goal of this protocol was to provide a set of clear, concise instructions to accompany video-based tutorials on how to measure the metabolic functions, such as the OCR and the ECAR, of HSPCs. With key modifications and additional recommendations for harvesting higher numbers of healthy LSK HSPCs, making them adherent to the surface of the well, as well as the optimization of the incubation time and drug concentration, this protocol will empower investigators to analyze glycolysis and mitochondrial respiration status of HSPCs as well as non-adherent hematopoietic cells during blood development and diseases.

Since the lifespan of most mature blood cells is short, the homeostasis of blood relies on the self-renewal and differentiation of a long-lived but rare population of hematopoietic stem cells (HSPCs)1. HSPCs are quiescent, but they are quick to proliferate and undergo differentiation upon stimulation to sustain demands of the blood system. As each HSPC cellular state requires a unique bioenergetic demand, the metabolic changes are key drivers of HSPC fate decisions. Therefore, the loss of metabolic plasticity, by altering the equilibrium between quiescence, self-renewal, and differentiation of HSPCs, often leads to myelo- or lympho-proliferative disorders. Together, the understanding of metabolic regulation of HSPC development is critical to uncover mechanisms underlying hematologic malignancies2,3,4,5.
Mitochondrial respiration and glycolysis generate ATP to drive intracellular reactions and produce the building blocks necessary for macromolecule synthesis. Since HSPCs have low mitochondrial mass compared to differentiated cells6 and they sustain quiescence in hypoxic bone marrow niches, HSPCs primarily rely on glycolysis. Activation of HSPCs enhances their mitochondrial metabolism that leads to the loss of quiescence and their subsequent entry into the cell cycle. Such metabolic plasticity of HSPCs allows the maintenance of the HSPC pool throughout adult life6,7,8,9,10,11,12. Therefore, it is critical to investigate their metabolic activities, such as the oxygen consumption rate (OCR; index of oxidative phosphorylation) and the extracellular acidification rate (ECAR; index of glycolysis) to analyze the HSPC activation and the health status. Both the OCR and the ECAR can be measured simultaneously, in real time, using an extracellular flux analyzer. However, the current method requires large numbers of cells and is optimized for adherent cells13. Since HSPCs cannot be isolated in large quantities from mice14, require sorting to obtain a pure population, are non-adherent cells15, and cannot be cultured overnight without avoiding differentiation16, it has been difficult to measure the OCR and the ECAR of HSPCs. Here, we provide a set of clear, step-by-step instructions to accompany video-based tutorials on how to measure metabolic respiration and glycolysis of few thousands murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs.

<table><tbody><tr><td>0.01% (w/v) poly-L-lysine solution</td><td>Sigma</td><td>P8920</td><td>Used for LSK attachment</td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Fisher Scientific</td><td>22-363-547</td><td>Used for cell filtration after bone crushing</td></tr><tr><td>Anti-Biotin MicroBeads</td><td>Miltenyi</td><td>130-090-485</td><td>Used for Lin- separation</td></tr><tr><td>Biotin Rat Anti-Mouse CD45R/B220 Clone RA3-6B2</td><td>BD Biosciences</td><td>553086</td><td>Used for Lin- separation</td></tr><tr><td>Biotin Rat Anti-Mouse CD5 Clone 53-7.3</td><td>BD Biosciences</td><td>553019</td><td>Used for Lin- separation</td></tr><tr><td>Biotin Rat Anti-Mouse CD8a Clone 53-6.7</td><td>BD Biosciences</td><td>553029</td><td>Used for Lin- separation</td></tr><tr><td>Biotin Rat Anti-Mouse Ly-6G and Ly-6C Clone RB6-8C5</td><td>BD Biosciences</td><td>553125</td><td>Used for Lin- separation</td></tr><tr><td>Biotin Rat Anti-Mouse TER-119/Erythroid Cells Clone TER-119</td><td>BD Biosciences</td><td>553672</td><td>Used for Lin- separation</td></tr><tr><td>CD117 (c-Kit) Monoclonal Antibody (2B8), APC</td><td>eBioscience</td><td>17-1171-83</td><td>Used for LSK sorting</td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>Falcon-Fischer Scientific</td><td>14-959-53A</td><td>Used in cell isolation</td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>Falcon-Fischer Scientific</td><td>14-432-22</td><td>Used in cell isolation</td></tr><tr><td>Falcon Round-Bottom Polypropylene Tubes</td><td>Falcon-Fischer Scientific</td><td>14-959-11A</td><td>Used for LSK sorting</td></tr><tr><td>Fetal Bovine Serum</td><td>Neuromics</td><td>FBS001-HI</td><td>Used in FACS buffer</td></tr><tr><td>Histopaque-1083</td><td>Sigma</td><td>10831</td><td>Used for ficoll gradient separation</td></tr><tr><td>L-glutamine 100x</td><td>Fisher Scientific</td><td>25-030-081</td><td>Used for the assay media</td></tr><tr><td>LS Column</td><td>Miltenyi</td><td>130-042-401</td><td>Used for Lin- separation</td></tr><tr><td>Ly-6A/E (Sca-1) Monoclonal Antibody (D7), PE-Cyanine7</td><td>eBioscience</td><td>25-5981-82</td><td>Used for LSK sorting</td></tr><tr><td>Murine Stem Cell Factor (SCF)</td><td>PeproTech</td><td>250-03-100UG</td><td>Used for the assay media</td></tr><tr><td>Murine Thrombopoietin (TPO)</td><td>PeproTech</td><td>315-14-100UG</td><td>Used for the assay media</td></tr><tr><td>PBS 1%</td><td>Fisher Scientific</td><td>SH3002802</td><td>Used for FACS buffer</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Fisher Scientific</td><td>15140122</td><td>Used for the assay media</td></tr><tr><td>Propidium Iodide</td><td>Fisher Scientific</td><td>P1304MP</td><td>Used for LSK sorting</td></tr><tr><td>Seahorse XFp Cell Culture Miniplate</td><td>Agilent Technologies</td><td>103025-100</td><td>Used for LSK seeding</td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>ThermoFisher</td><td>11360070</td><td>Used for the assay media</td></tr><tr><td>Streptavidin eFluor 450 Conjugate</td><td>eBioscience</td><td>48-4317-82</td><td>Used for LSK sorting</td></tr><tr><td>XF Calibrant</td><td>Agilent Technologies</td><td>100840-000</td><td>Used for cartridge equilibration</td></tr><tr><td>XF media</td><td>Agilent Technologies</td><td>103575-100</td><td>Used for the assay media</td></tr><tr><td>XFp Glycolysis Stress Test Kit</td><td>Agilent Technologies</td><td>103017100</td><td>Drugs for glycolysis stress test</td></tr><tr><td>XFp Mitochondrial Stress Test Kit</td><td>Agilent Technologies</td><td>103010100</td><td>Drugs for mitochondrial stress test</td></tr><tr><td>XFp Sensor Cartridge</td><td>Agilent Technologies</td><td>103022-100</td><td>Used for glycolysis and mitochondrial stress test</td></tr></tbody></table>

analysis of hematopoietic stem progenitor cell metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Our extraction method allowed us to harvest up to ~80,000 LSK HSPCs per mouse. The viability and numbers of LSK cells were improved with our method, because we: (1) combined bone marrow from upper and lower limbs, hip bones, sternum, rib cage, and spine, (2) avoided using red cell lysis buffer that would have increased cell-death and clumping, (3) used the density gradient medium separation of mono-nucleated cells, and (4) avoided using pre-chilled buffer that would have caused the loss of cells-of-interest in clumps.
Although extracellular flux analysis has been traditionally used for adherent cells, our use of the PLL coating of wells, followed by centrifugation of cells on it, facilitated adherence of LSK HSPCs to the surface of the well. This allowed us to measure the extracellular flux, and thus metabolic health of LSK HSPCs. Considering the limited number of cells that can be harvested from a mouse and the long duration of the protocol for their isolation, our use of the analyzer with its 8 well format has emerged as the most cost-effective and feasible solution (Figure 1).
Cells use glycolysis and mitochondrial respiration to replenish their energy requirements and to produce intermediates needed for their proliferation and growth19. The hexokinase enzyme converts glucose in glucose-6-phosphate and that is subsequently transformed into pyruvate20. Pyruvate can then be processed into lactate and is exported from the cell with protons21. ECAR measures the acidification of the media and is thus an indicator of glycolysis. Pyruvate can also be transported into the mitochondria and transformed in acetyl coenzyme A (CoA). Acetyl CoA enters the TCA cycle, which provides energy intermediates to drive the electron movements of the Electron Transport Chain (ETC) and generates a proton gradient in the mitochondrial inter-membrane space22. Oxygen acts as the final electron acceptor, and protons move back to the mitochondrial matrix through the ATP synthase complex while generating ATP23. OCR measures the oxygen consumption and it is therefore used to quantify the mitochondrial respiration.
In order to analyze the OCR and the ECAR in basal and stressed conditions, we used sequential injection of drugs that interfere with glycolysis and mitochondrial respiration. We used glucose and 2-deoxyglucose (2-DG), a glucose analogue, to initiate and block glycolysis respectively24. We used Rotenone (a complex I-specific inhibitor of the ETC), antimycin A (a complex III-specific inhibitor of the ETC), oligomycin (inhibitor of ATP synthase), and the uncoupling agent carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) to block specific events of the ETC25. We titrated such reagents to find the optimal concentration for LKS HSPCs (Figure 2A,B).
To perform the glycolysis stress test, we cultured the LSK HSPCs in a glucose/pyruvate deprived media (as recommended by the manufacturer), or in glucose/pyruvate containing media. As expected, we found the basal level of the ECAR was higher for LSK HSPCs cultured in glucose/pyruvate+ media compared to LSK HSPCs cultured in glucose/pyruvate- media. The first injection with glucose did not change the basal level of ECAR for LSK HSPCs cultured in glucose/pyruvate+ media while it boosted glycolysis in LSK HSPCs cultured in glucose/pyruvate- media. However, the basal level of the ECAR, after the injection with glucose, remained lower compared to the glucose/pyruvate+ group. The second injection with oligomycin, which could block the production of ATP through oxidative phosphorylation, activated the glycolysis at its maximum level of LSK HSPCs in glucose/pyruvate+ media, but it did not affect the glucose/pyruvate- group. The last injection with the glucose analogue 2-DG returned the ECAR to its non-glycolytic level (Figure 2C).
For the mitochondrial stress test, we measured the basal level of OCR of LSK HSPCs in glucose/pyruvate+ media. We first injected oligomycin that initially hyperpolarized the mitochondrial membrane, prevented more proton pumping through the ETC complexes, and thus reduced the rate of mitochondrial respiration. The second injection of FCCP ionophores pushed ETC and OCR levels to their maximum as cells tried to recover the mitochondrial membrane potential. The final injection with other two of the ETC inhibitors (antimycin A and rotenone) caused the complete stop of mitochondrial respiration and thus the OCR reverted to its minimum level (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60234/60234fig01.jpg" /> 
Figure 1: Schema demonstrating isolation of LineagenegSca1+c-Kit+ (LSK) hematopoietic stem progenitor cells from mouse bone marrow. Bone marrow is extracted from bones and mononuclear cells (MNCs) are isolated through density gradient medium gradient separation. Next, cells are incubated with biotinylated Lineage+ antibodies and streptavidin-conjugated magnetic beads to elute Lineage negative (Lin-) cells following their magnetic separation. Lin- cells are subsequently incubated with LSK antibodies and LSK cells isolated by cell sorting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60234/60234fig01large.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/60234/60234fig02.jpg" /> 
Figure 2: Extracellular flux analyses of murine LineagenegSca1+c-Kit+ (LSK) hematopoietic stem progenitor cells. (A,B) Mechanistic description of drugs utilized for extracellular flux analyses during glycolysis and mitochondrial respiration. (C) Representative results of glycolysis stress test on murine LSK HSPCs in presence or absence of glucose/pyruvate in the media. (D) Representative results of mitochondrial stress test on murine LSK HSPCs. Error bars represent the standard deviation of the mean (S.D.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60234/60234fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Stock</td>
			<td>Final concentration</td>
			<td>Volume for 30 mL</td>
		</tr>
		<tr>
			<td>Complete media</td>
			<td></td>
			<td></td>
			<td>28.691 mL</td>
		</tr>
		<tr>
			<td>P/S</td>
			<td>100x</td>
			<td>0.5x</td>
			<td>150 &#181;L</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>200 mM</td>
			<td>2 mM</td>
			<td>300 &#181;L</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>100 mM</td>
			<td>1 mM</td>
			<td>300 &#181;L</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1 M/180.2 mg/mL</td>
			<td>3 mg/mL</td>
			<td>499.4 &#181;L</td>
		</tr>
		<tr>
			<td>TPO</td>
			<td>100 &#181;g/mL</td>
			<td>100 ng/mL</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>100 &#181;g/mL</td>
			<td>100 ng/mL</td>
			<td>30 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Contents and preparation of the complete XF media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Basal</td>
			<td>Oligomycin (2 &#181;M)</td>
			<td>FCCP (1.5 &#181;M)</td>
			<td>R/A (0.5 &#181;M)</td>
		</tr>
		<tr>
			<td>Repetition</td>
			<td>3 times</td>
			<td>3 times</td>
			<td>3 times</td>
			<td>3 times</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>3 min</td>
			<td>3 min</td>
			<td>3 min</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>0 min</td>
			<td>0 min</td>
			<td>0 min</td>
			<td>0 min</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>4 min</td>
			<td>4 min</td>
			<td>4 min</td>
			<td>4 min</td>
		</tr>
	</tbody>
</table>
Table 2: Injection protocol for the mitochondrial stress test.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Basal</td>
			<td>Glucose (10 mM)</td>
			<td>Oligomycin (2 &#181;M)</td>
			<td>2-DG (50 mM)</td>
		</tr>
		<tr>
			<td>Repetition</td>
			<td>3 times</td>
			<td>3 times</td>
			<td>3 times</td>
			<td>3 times</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>3 min</td>
			<td>3 min</td>
			<td>3 min</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>0 min</td>
			<td>0 min</td>
			<td>0 min</td>
			<td>0 min</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>7 min</td>
			<td>7 min</td>
			<td>7 min</td>
			<td>7 min</td>
		</tr>
	</tbody>
</table>
Table 3: Injection protocol for the glycolysis stress test.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Port</td>
			<td>Drug</td>
			<td>Final well concentration (&#181;M)</td>
			<td>Stock solution volume (&#181;L)</td>
			<td>Media volume (&#181;L)</td>
			<td>Port solution (&#181;M)</td>
			<td>Volume added to port (&#181;L)</td>
		</tr>
		<tr>
			<td>Port A</td>
			<td>Oligomycin</td>
			<td>2</td>
			<td>100</td>
			<td>181.25</td>
			<td>16</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Port B</td>
			<td>FCCP</td>
			<td>1.5</td>
			<td>100</td>
			<td>270.4</td>
			<td>13.5</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Port C</td>
			<td>Rotenone/antimycin A</td>
			<td>0.5</td>
			<td>60</td>
			<td>240</td>
			<td>5</td>
			<td>25</td>
		</tr>
	</tbody>
</table>
Table 4: Dilution metrics to obtain optimum drug concentration for mitochondrial stress test in each well of the analyzer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Port</td>
			<td>Drug</td>
			<td>Final well concentration</td>
			<td>Stock solution volume (&#181;L)</td>
			<td>Media volume (&#181;L)</td>
			<td>Port solution</td>
			<td>Volume added to port (&#181;L)</td>
		</tr>
		<tr>
			<td>Port A</td>
			<td>Glucose</td>
			<td>10 mM</td>
			<td>300</td>
			<td>75</td>
			<td>80 mM</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Port B</td>
			<td>Oligomycin</td>
			<td>2 &#181;M</td>
			<td>108</td>
			<td>192</td>
			<td>18 &#181;M</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Port C</td>
			<td>2-DG</td>
			<td>50 mM</td>
			<td>300</td>
			<td>0</td>
			<td>500 mM</td>
			<td>25</td>
		</tr>
	</tbody>
</table>
Table 5: Dilution metrics to obtain optimum drug concentration for glycolysis stress test in each well of the analyzer.

Watch this Scientific Journal Video about Analysis of Hematopoietic Stem Progenitor Cell Metabolism at JoVE.com

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

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6.8K Views.  Nationwide Children's Hospital. Metabolische veranderingen in hematopoietische stam- en voorlopercellen stellen hen in staat om over te stappen van hun rustige toestand naar een differentiatietoestand om eisen van bloedvorming te ondersteunen. Verandering in metabole plasticiteit van hematopoietische stam- en voorlopercellen leidt tot hematologische aandoeningen. Ons protocol zal helpen meten van de metabole regulatie en de gezondheid van hematopoietische stam- en voorlopercellen tijdens de bloedontwikkeling en ziekten....

Video: Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Immunology and Infection

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Cancer Research

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Biology

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

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.

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 ...

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 ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

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