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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 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.
Hematopoiesis is the process by which various types of mature blood cells with highly specialized functions are formed from HSCs1. HSCs are capable of self-renewal and differentiation into various multipotent and lineage-specific progenitor populations. These progenitors ultimately produce cells of lymphoid, myeloid, erythroid, and megakaryocyte lineages. To maintain their self-renewal capacity, HSCs remain largely quiescent and, like other tissue stem cells, are believed to rely on glycolysis rather than mitochondrial OxPHOS for ATP production2,3. Entry into the cell cycle leads to enhanced respiration and OxPHOS, resulting in elevated levels of reactive oxygen species (ROS), which are detrimental to HSC maintenance and function3. Repeated cell division thus may lead to reduced self-renewal capacity of HSCs and ultimately to their exhaustion.
In adult hematopoiesis, HSCs primarily undergo asymmetric cell division, during which one of the daughter cells retains HSC potential and continues to rely on glycolytic metabolism. The other daughter cell becomes a primitive progenitor cell that loses self-renewal capacity but proliferates and eventually gives rise to differentiated functional hematopoietic cells4. When HSCs differentiate to produce downstream progenitors, a switch from glycolysis to mitochondrial metabolism is thought to occur to supply the energy and building blocks needed to support this rapid transition5, as suggested by the observations that HSCs possess inactive mitochondrial mass6,7,8,9. In contrast, mitochondrial activity (indicated by linked ROS levels) is much higher in lineage-committed progenitors than in HSCs9,10,11. Metabolic changes that occur during the earliest step of hematopoiesis thus suggest a direct and crucial role of mitochondria in regulating HSC fate.
Dysfunctional mitochondria present under various stress conditions, such as aging, cancer, diabetes, and obesity12, can interfere with HSC self-renewal capacity, inducing an imbalance in HSC/progenitor differentiation by producing excessive amounts of ROS, impairing ATP production, and/or by altering other metabolic processes9,12,13. Perturbations in metabolic homeostasis in HSC/progenitor differentiation could significantly impact hematopoiesis, potentially contributing to the development of hematologic abnormalities13. Given the critical influences of glycolysis and mitochondrial OxPHOS on HSC stemness and differentiation, it is of interest to investigate both metabolic parameters under normal and stress conditions. Valuable insights into the glycolytic and mitochondrial function of HSCs and progenitor cells can be gained by assessing their ECAR and OCR, which are indicators of cellular glycolysis and mitochondrial respiration, respectively.
The Seahorse extracellular flux analyzer is a powerful apparatus equipped with two probes per well to simultaneously measure ECAR and OCR in live cells and thus can be used to assess cellular bioenergetics in real time, in response to various substrates or inhibitors. The assay cartridge used with the analyzer contains injection ports to hold up to four drugs for automated injection during the assay. A scheme of a typical glycolysis stress test is shown in Figure 1A. The assay starts with the measurement of ECAR of cells, incubated in glycolysis stress test medium containing glutamine but not glucose or pyruvate. This represents acidification occurring due to non-glycolytic activities of the cells and is reported as non-glycolytic acidification. This is followed by the injection of glucose at a saturating concentration. Via glycolysis, glucose in the cell is converted into pyruvate, which is further metabolized in the cytoplasm to produce lactate, or in mitochondria to produce CO2 and water.
Conversion of glucose to lactate causes net production and subsequent release of protons into the extracellular medium, resulting in a rapid increase in the ECAR14,15,16. This glucose-stimulated change in ECAR is reported as glycolysis under basal conditions. The second injection consists of oligomycin (an inhibitor of ATP synthase, a.k.a. complex V17), which inhibits mitochondrial ATP production. During oligomycin-mediated OxPHOS inhibition, cells maximally upregulate glycolysis to meet their energetic demands. This causes a further increase in ECAR, revealing the maximum glycolytic capacity of the cells. The difference between the maximum glycolytic capacity and basal glycolysis is referred to as glycolytic reserve. Finally, 2-DG is injected, which causes a significant drop in ECAR, usually close to non-glycolytic acidification levels. 2-DG is a glucose analog that competitively binds to hexokinase, resulting in inhibition of glycolysis18. Thus, the 2-DG-induced decrease in ECAR further confirms that glycolysis is indeed the source of ECAR observed after glucose and oligomycin injections.
Figure 1B displays the schematic for a typical mitochondrial stress test. The assay starts with baseline OCR measurement of the cells, incubated in mitochondrial stress test medium containing glucose, glutamine, and pyruvate. Following basal OCR measurements, oligomycin is injected in this assay, which inhibits complex V, thereby reducing electron flow through the electron transport chain (ETC)17. Consequently, OCR is reduced in response to oligomycin injection, and this decrease in OCR is linked to mitochondrial ATP production. The second injection consists of carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), a protonophore and an uncoupler of mitochondrial OxPHOS17. FCCP collapses the mitochondrial proton gradient by allowing the flow of protons across the mitochondrial inner membrane. Because of the FCCP injection, electron flow through the ETC is derepressed, and complex IV consumes oxygen at the maximal level. The difference between maximal OCR and basal OCR is referred to as the spare respiratory capacity, which is a measure of the cell's ability to respond to increased energy demand under stress conditions. Finally, a mixture of two ETC inhibitors (rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor17) is injected, which completely shuts down electron flow, and OCR decreases to a low level. OCR measured after rotenone and antimycin A injection corresponds to non-mitochondrial OCR driven by other processes inside the cells. Non-mitochondrial OCR enables the calculation of basal respiration, proton leak, and maximal respiration.
Basal respiration represents the difference between baseline OCR and non-mitochondrial OCR. Proton leak refers to the difference between OCR after oligomycin injection and non-mitochondrial OCR. Maximal respiration represents the difference between OCR after FCCP injection and non-mitochondrial OCR. Coupling efficiency is calculated as the percentage of ATP production rate to basal respiration rate. This method paper provides a detailed protocol to measure ECAR and OCR in lineage-negative HSPCs using the Seahorse XFe96 extracellular flux analyzer. This protocol describes approaches to isolate mouse lineage-negative HSPCs, explains the optimization of cell-seeding density and concentrations of various drugs used in extracellular flux assays, and describes drug treatment strategies.
All vertebrate animal experiments were approved by and performed in accordance with the regulations of the University of Michigan Committee on Use and Care of Animals.
1. Day before the assay (Total time: ~10 min)
2. Day of the assay (Total time: ~9 h 30 min)
NOTE: The total time indicated above includes cumulative durations of steps 2.1-2.4 in addition to either step 2.5 or step 2.6.
Using this protocol, the cell number and the concentrations of various OxPHOS-targeting drugs (used in the extracellular flux assays) were optimized to measure ECAR and OCR of HSPCs isolated from 24-week-old female C57BL/6 mice. First, the glycolysis stress test was performed to optimize cell number and oligomycin concentration. A varying number of HSPCs per well ranging from 5 × 104 to 2.5 × 105 were used in this assay. As shown in Figure 2A and
This method paper describes an optimized protocol for the assessment of cellular bioenergetics (glycolysis and OxPHOS) in mouse HSPCs using the Seahorse extracellular flux analyzer. This device is a powerful tool that simultaneously measures the ECAR and OCR of live cells, which are metrics of glycolysis and mitochondrial respiration, respectively. Thus, it can be used to assess the cellular bioenergetics in real time. Further, the 96-well microplate-based platform offers high-throughput quantification with high sensitiv...
The authors declare that there is no conflict of interest.
Work in Lombard laboratory is supported by the NIH (NIGMS R01GM101171, NIEHS R21ES032305), DoD (CA190267, CA170628, NF170044, and ME200030), and Glenn Foundation for Medical Research. Work in Li laboratory is supported by NIH (NHLBI 5R01HL150707).
Name | Company | Catalog Number | Comments |
0.2 μm filter | Corning | 430626 | Used to filter-sterilize the assay media |
100 mM sodium pyruvate | Life Technologies | 11360-070 | Component of mitochondrial stress test assay medium |
15 mL conical Falcon tubes | Corning | 352096 | Used during HSPCs harvest and to prepare assay drug solutions |
200 mM L-glutamine | Life Technologies | 25030-081 | Component of glycolysis stress test and mitochondrial stress test assay media |
2-Deoxy-D-glucose (2-DG) | Sigma-Aldrich | D8375 | 3rd drug injection during glycolysis stress test |
5x Enrichment buffer (MojoSort) | Biolegend | 480017 | Used for washings during HSPCs harvest |
Ammonium chloride (NH4Cl) | Fisher Scientific | A661-3 | Component of ACK lysis buffer |
Antimycin A | Sigma-Aldrich | A8674 | 3rd drug injection during mitochondrial stress test |
Bio-Rad DC protein assay kit | Bio-Rad | 500-0112 | Used as per manufacturer's instructions |
Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) | Sigma-Aldrich | C2920 | 2nd drug injection during mitochondrial stress test |
Cell-Tak | Corning | 354240 | Cell adhesive. Used for coating cell microplates |
Countes 3 Automated Cell Counter | ThermoFisher Scientific | For cell counting | |
EDTA | Fisher Scientific | O2793-500 | Component of ACK lysis buffer and RIPA lysis buffer |
Falcon 70 μm filter | Fisher Scientific | 08-771-2 | Used as cells strainer during HSPCs harvest |
Gibco Fetal bovine serum (FBS) | Fisher Scientific | 26400044 | Used to prepare assay buffer during HSPCs harvest |
Gibco HBSS | Fisher Scientific | 14175095 | Used to prepare assay buffer during HSPCs harvest |
Glucose | Sigma-Aldrich | G7528 | Component of mitochondrial stress test assay medium and first injection of glycolysis stress test |
Oligomycin | Sigma-Aldrich | O4876 | 2nd drug injection during glycolysis stress test and 1st drug injection during mitochondrial stress test |
PBS | Life Technologies | 10010-049 | Used to wash cells after assay for protein quantification |
Potassium bicarbonate (KHCO3) | Fisher Scientific | P235-500 | Component of ACK lysis buffer |
Protease Inhibitor Cocktail (PIC) | Roche | 11836170001 | Supplied as tablets. One tablet was dissolved in 10 mL of RIPA buffer to make 1x PIC. |
Rat biotin antimouse-B220, Clone ID: RA3-6B2 | Biolegend | 103203 | Used for lineage depletion during HSPCs harvest |
Rat biotin antimouse-CD2, Clone ID: RM2-5 | Biolegend | 100103 | Used for lineage depletion during HSPCs harvest |
Rat biotin antimouse-CD3, Clone ID: 17A2 | Biolegend | 100243 | Used for lineage depletion during HSPCs harvest |
Rat biotin antimouse-CD5, Clone ID: 53-7.3 | Biolegend | 100603 | Used for lineage depletion during HSPCs harvest |
Rat biotin antimouse-CD8, Clone ID: 53-6.7 | Biolegend | 100703 | Used for lineage depletion during HSPCs harvest |
Rat biotin antimouse-Gr-1, Clone ID: RB6-8C5 | Biolegend | 108403 | Used for lineage depletion during HSPCs harvest |
Rat biotin antimouse-Ter-119, Clone ID: TER-119 | Biolegend | 116203 | Used for lineage depletion during HSPCs harvest |
Rotenone | Sigma-Aldrich | R8875 | 3rd drug injection during mitochondrial stress test |
Seahorse XFe96 extracellular flux analyzer | Seahorse Biosciences now Agilent | For ECAR and OCR measurments in real time. | |
Sodium bicarbonate | Sigma-Aldrich | S5761 | Used to make Cell-adhesive solution for microplate coating |
Sodium chloride (NaCl) | Fisher | BP358 | Component of RIPA lysis buffer |
Sodium deoxycholate | Sigma-Aldrich | D6750 | Component of RIPA lysis buffer |
Sodium Fluoride (NaF) | Sigma-Aldrich | S7920 | Component of RIPA lysis buffer |
Sodium hydroxide (NaOH) | Sigma-Aldrich | S8045 | Prepared 1 N solution. Used for pH normalization |
Streptavidin Nanobeads (MojoSort) | Biolegend | 480015 | Used for lineage depletion during HSPCs harvest |
Tris-HCl | Fisher | BP153 | Component of RIPA lysis buffer |
XF base medium | Agilent | 102353-100 | base medium used to prepare glycolysis stress test and mitochondrial stress test assay media |
XF prep station | Seahorse Biosciences | Used for non-CO2 37 °C incubations | |
XFe96 extracellular FluxPak | Agilent | 102416-100 or 102601-100 | Includes assay cartridges with utility plates, loading guide flats for loading drugs onto the assay cartridge, XF calibrant solution, and XF cell culture microplate |
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