Jan Van den Bossche received a Junior Postdoc grant from the Netherlands Heart Foundation (2013T003) and a VENI grant from ZonMW (91615052). Menno de Winther is an Established Investigator of the Netherlands Heart Foundation (2007T067), is supported by a Netherlands Heart Foundation grant (2010B022) and holds an AMC-fellowship. We also acknowledge the support from the Netherlands Cardiovascular Research Initiative, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development and the Royal Netherlands Academy of Sciences for the GENIUS project &#8220;Generating the best evidence-based pharmaceutical targets for atherosclerosis&#8221; (CVON2011-19). We acknowledge Seahorse Bioscience for making open access publication possible. The authors thank Riekelt Houtkooper and Vincent de Boer (Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands) for all previous assistance with the Seahorse analyzer.

1. Culturing and Polarizing Bone Marrow-derived Macrophages
<ol>
	<li>Day 0: Isolate femur and tibia bones from 6-10 week old mice of interest (C57Bl/6 in this assay).</li>
	<li>Remove muscle tissue from the bones and place the bones in a 9 cm petri-dish filled with ice-cold PBS.</li>
	<li>Transfer the bones to a 9 cm Petri dish filled with 70% ethanol and gently shake the plate for 30 sec.</li>
	<li>Transfer the bones to a fresh 9 cm Petri dish filled with ice-cold sterile PBS.</li>
	<li>Cut the femur and tibia at both ends with sterile scissors.</li>
	<li>Flush the bones with 10 ml ice-cold sterile PBS using a 10 ml syringe and a 25 G needle. Collect the bone marrow in a 50 ml tube.</li>
	<li>Transfer the bone marrow cells to a new 50 ml tube using a 23 G needle.</li>
	<li>Repeat this step with a 25 G needle. Note: These steps are needed to remove cell clumps and bone residues.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the cells in 40 ml cell culture medium (RPMI-1640 plus 2 mM L-glutamine, 10% FCS, penicillin (100 U/ml), streptomycin (100 &#181;g/ml) and 15% filtered L-929 cell (ATCC,CCL-1)-conditioned medium containing M-CSF (or alternatively 10 ng/ml recombinant M-CSF instead of L-929 cell-conditioned medium). 
	Note: The generation of L-929 cell-conditioned medium was detailed earlier in this journal in 2013.11</li>
	<li>Culture the cells from one mouse in two 15 cm Petri dishes (do not use cell culture-treated plates since macrophage will not detach from this plastic) in 20 ml culture medium per plate in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Add 10 ml fresh cell culture medium on day 3, without removing the 20 ml older cell culture medium.</li>
	<li>Replace the old medium of each plate with 20 ml fresh culture medium on day 6. Doing this, non-adherent cells will also be removed.</li>
	<li>Day 8:
	<ol>
		<li>Detach cells by incubating them 5 min at 37 &#176;C in 10 ml citrate saline (135 mM potassium chloride plus 15 mM sodium citrate in H2O, autoclaved) and wash with 10 ml PBS. Count mature bone marrow-derived macrophages (BMDM) on day 8 using a cell counter.</li>
		<li>Verify the formation of mature (CD11b+ F4/80+) BMDM by visual inspection or by flow cytometry. Block 105 cells with 1/100 anti-CD16/CD32 in PBS for 20 min in 100 &#181;l PBS, incubate with 1/200 anti-CD11b-FITC plus 1/200 anti-F4/80-APC-Cy7 at RT, wash and analyze by flow cytometry. 
		Note: Culturing mouse bone marrow-derived macrophages has been published in detail earlier in this journal by Ying et al.11.</li>
	</ol>
	</li>
	<li>Seed 50,000 cells (optimal cell counts need to be optimized) per well in a cell culture microplate in 100 &#181;l culture medium. Do not seed cells in background correction wells (A1, A12, H1, H12) and put medium only (no cells) in these wells. Tip: Hold the pipette at an angle about halfway down the side of the wells for most homogeneous cell layer.</li>
	<li>Allow the cells to settle at RT in a cell culture hood for 1 hr. This will promote even cell distribution and reduce edge effects. Monitor adherence using a microscope and culture in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Three hr after plating, stimulate cells for 24 hr with 10 ng/ml LPS, or 20 ng/ml recombinant mouse IL-4 to generate M1 or M2 macrophages, respectively. Include untreated na&#239;ve (M0) macrophages as a control. Assess proper M1 and M2 macrophage polarization by measuring LPS-induced secretion of IL-6, IL-12, TNF (ELISA) and IL-4-induced arginase-1 activity and/or MMR (CD206) and MGL (CD301) surface expression (flow cytometry) as described earlier3,11. 
	Note: At this point, cells can be (pre)-treated with pharmacological compounds of interest or macrophages can be stimulated with other factors. Since, the variation between separate wells might be substantial, it is advisable to use at least 4 and ideally 6 wells per condition.</li>
</ol>
2. Preparation of the Extracellular Flux Assay
<ol>
	<li>Hydrate the sensor cartridge one day before running the assay:
	<ol>
		<li>Place the sensor cartridge upside down next to the utility plate.</li>
		<li>Fill each well of the utility plate with 200 &#181;l of calibrant solution and lower the sensor cartridge onto the utility plate submerging the sensors in the calibrant solution.</li>
		<li>Verify the calibrant solution level is high enough to keep the sensors submerged and place in a non-CO2 37 &#176;C incubator O/N.</li>
	</ol>
	</li>
	<li>Prepare assay medium on the day of the assay as follows:
	<ol>
		<li>Warm 50 ml XF base medium to 37 &#176;C.</li>
		<li>Add 500 &#181;l 200 mM L-glutamine to get a 2 mM final concentration.</li>
		<li>Adjust pH to 7.4 using 1 N NaOH and sterilize with a 0.2 &#181;M filter and keep the assay medium at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Gently remove the cell culture medium from the polarized macrophages and store it at -20 &#176;C for future use (e.g. ELISA to examine proper macrophage polarization). Wash the cells with 100 &#181;l assay medium, add 180 &#181;l assay medium per well and verify under the microscope that cells were not washed away. Place the cell culture plate in a 37 &#176;C incubator without CO2 for 1 hr prior to the assay run.</li>
	<li>Prepare 10 x injection compound mixtures A till D as described in Table 1, warm to 37 &#176;C, adjust the pH to 7.4 and filter sterilize.
	<ol>
		<li>Make all compounds in these mixtures at 10x the final concentration in the cell culture wells and optimize these concentrations for each cell type.</li>
	</ol>
	</li>
	<li>Load the sensor cartridge using the provided loading guides with indicated volumes (Table 1) of the prepared 10x injection compound mixtures in ports A, B, C and D, respectively.</li>
</ol>
<table border="0">
	<tbody>
		<tr>
			<td>Mixture/Injection</td>
			<td>Compounds&#160;</td>
			<td>Volume added to get 10x mixture (&#181;l)</td>
			<td>Volume Assay Medium (ml)</td>
			<td>Volume injected during run (&#181;l)</td>
			<td>Final concentration in the assay</td>
		</tr>
		<tr>
			<td>A</td>
			<td>&#160;2.5 M (45%) glucose&#160;</td>
			<td>300</td>
			<td>2.7</td>
			<td>20</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>B</td>
			<td>5 mM oligomycin A (OM)&#160;</td>
			<td>9</td>
			<td>3.0</td>
			<td>22</td>
			<td>1.5 &#181;M</td>
		</tr>
		<tr>
			<td>C</td>
			<td>5 mM FCCP&#160;</td>
			<td>9</td>
			<td>2.7</td>
			<td>25</td>
			<td>1.5 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td>100 mM Sodium Pyruvate solution&#160;</td>
			<td>300</td>
			<td></td>
			<td></td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>D</td>
			<td>5 mM antimycin A (AA)</td>
			<td>15</td>
			<td>3.0</td>
			<td>28</td>
			<td>2.5 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td>5 mM rotenone (Rot)&#160;</td>
			<td>7.5</td>
			<td></td>
			<td></td>
			<td>1.25 &#181;M</td>
		</tr>
	</tbody>
</table>
Table 1. Injection mixtures.
3. Bioenergetic Characterization of Polarized M1 and M2 BMDM using a Extracellular Flux Analyzer
<ol>
	<li>Create an assay template with the assay wizard with 2 minute MIX and 3 minute MEASURE times and (at least) 3 mix and rate measurements loops before each of the 4 injections (Table 1) and after the last injection. 
	Note: These concentrations are valid for bone marrow-derived macrophages and Raw264.7 macrophage cell lines, but should be optimized for each cell type. Adding pyruvate in the FCCP mixture is needed to fuel maximal respiration.</li>
	<li>Start the run by pushing the start bottom and follow the instructions of the apparatus. First load the cartridge plate with the hydrated probes to allow calibration by apparatus and next load the cell plate.</li>
	<li>After the assay, carefully discard all assay medium and store the analyzed cell culture microplate at -20 &#176;C for future cell normalization using the cell proliferation assay kit as described in detail by the supplier.
	<ol>
		<li>Briefly, prepare a 1x cell-lysis buffer by diluting component B 20-fold in distilled water and dilute compound A 400-fold in the 1x cell-lysis buffer. Add 200 &#181;l per well, incubate 5 min at RT and measure fluorescence with ~180 nm excitation and ~520 nm emission maxima. 
		Note: When working with non-adherent cells or if poor adherence is a concern, the direct cell proliferation kit can be used as an alternative since this protocol does not require discarding all assay medium. Yet, this direct protocol is slightly less sensitive compared to the protocol used in this lab.</li>
	</ol>
	</li>
	<li>After fluorescence measurement, normalize the cell count in each well as a ratio in which the average cell count of all na&#239;ve macrophage wells is set at 1. 
	Note: Protein quantification (e.g. using a BCA assay) could be used as an alternative way to normalize cells counts. Yet, in our hands this assay was less sensitive compared to the cell proliferation assay kit.</li>
</ol>

<ol>
	<li>Murray, P. J., 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=Macrophage+activation+and+polarization:+nomenclature+and+experimental+guidelines.">Macrophage activation and polarization: nomenclature and experimental guidelines.</a> Immunity. 41, 14-20 (2014).</li><li>Hoeksema, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN+gamma+Priming+of+Macrophages+Represses+a+Part+of+the+Inflammatory+Program+and+Attenuates+Neutrophil+Recruitment.">IFN gamma Priming of Macrophages Represses a Part of the Inflammatory Program and Attenuates Neutrophil Recruitment.</a> J Immunol. , (2015).</li><li>Vanden Bossche, J., 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=Pivotal+Advance+Arginase+1+independent+polyamine+production+stimulates+the+expression+of+IL+4+induced+alternatively+activated+macrophage+markers+while+inhibiting+LPS-induced+expression+of+inflammatory+genes.">Pivotal Advance Arginase 1 independent polyamine production stimulates the expression of IL 4 induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes.</a> Journal of leukocyte biology. 91, 685-699 (2012).</li><li>Pearce, E. L., Pearce, 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=Metabolic+pathways+in+immune+cell+activation+and+quiescence.">Metabolic pathways in immune cell activation and quiescence.</a> Immunity. 38, 633-643 (2013).</li><li>Yang, L., 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=PKM2+regulates+the+Warburg+effect+and+promotes+HMGB1+release+in+sepsis.">PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis.</a> Nature communications. 5, 4436 (2014).</li><li>Freemerman, A. J., 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+reprogramming+of+macrophages+glucose+transporter+1+(GLUT1)+mediated+glucose+metabolism+drives+a+proinflammatory+phenotype.">Metabolic reprogramming of macrophages glucose transporter 1 (GLUT1) mediated glucose metabolism drives a proinflammatory phenotype.</a> The Journal of biological chemistry. 289, 7884-7896 (2014).</li><li>Tavakoli, S., Zamora, D., Ullevig, S., Asmis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+profiles+diverge+during+macrophage+polarization+implications+for+the+interpretation+of+18F+FDG+PET+imaging+of+atherosclerosis.">Bioenergetic profiles diverge during macrophage polarization implications for the interpretation of 18F FDG PET imaging of atherosclerosis.</a> J Nucl Med. 54, 1661-1667 (2013).</li><li>Vats, D., 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=Oxidative+metabolism+and+PGC+1beta+attenuate+macrophage+mediated+inflammation.">Oxidative metabolism and PGC 1beta attenuate macrophage mediated inflammation.</a> Cell metabolism. 4, 13-24 (2006).</li><li>Huang, S. 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=Cell+intrinsic+lysosomal+lipolysis+is+essential+for+alternative+activation+of+macrophages.">Cell intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.</a> Nature immunology. 15, 846-855 (2014).</li><li>Tannahill, G. 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=Succinate+is+an+inflammatory+signal+that+induces+IL+1beta+through+HIF+1alpha.">Succinate is an inflammatory signal that induces IL 1beta through HIF 1alpha.</a> Nature. 496, 238-242 (2013).</li><li>Ying, W., Cheruku, P. S., Bazer, F. W., Safe, S. H., Zhou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+macrophage+polarization+using+bone+marrow+derived+macrophages.">Investigation of macrophage polarization using bone marrow derived macrophages.</a> J Vis Exp. (76), (2013).</li><li>Hoeksema, M. 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Free access to this article is sponsored by Seahorse Bioscience.

The aim of our laboratory is to understand how macrophage polarization and function can be influenced with the ultimate goal to improve outcome of atherosclerosis and other inflammatory conditions12. To assess macrophage polarization, one typically measures the LPS(+IFN&#947;)-induced and IL-4-induced gene expression of M1 and M2 marker genes (qPCR), determines IL-6, IL-12, TNF and NO secretion (by ELISA and Griess reaction) and examines IL-4-induced CD71, CD206, CD273 and CD301 surface expression (by flow cytometry) and arginase activity3,13. Additionally, apoptosis assays (by Annexin-V/PI staining), phagocytosis assays (with fluorescent latex beads and/or pHrodo E. coli) and foam cell formation assays (by DiI-oxLDL uptake, LipidTOX neutral lipid staining) can be performed to evaluate macrophage functionality14. Additionally, extracellular flux analysis can be performed to measure bioenergetics profiles of polarized macrophages as a new and alternative functional readout.
This manuscript shows that macrophage polarization induces metabolic reprogramming with LPS-induced macrophages switching to increased glycolysis as their energy source. Conversely, IL-4-induced M2 macrophages use mitochondrial oxidative phosphorylation as the main source of ATP. Importantly, metabolic reprogramming not only provides energy for the particular requirements of M1 and M2 polarized macrophages. Indeed, metabolic changes affect metabolite concentrations that are direct regulators of the macrophage phenotype 10,15,16. Hence, macrophage metabolism is not only a characteristic (as shown here in Figure 2) but also a driver of distinct macrophage polarization states and this new knowledge supported the rapid growth of the immunometabolism research area during the last couple of years.
However, robust measurements of metabolic profiles in macrophages remained difficult and had their limitations. In this study, an extracellular flux analyser is applied to robustly measure glycolysis, glycolytic reserve, maximal glycolytic capacity, non-glycolytic acidification, basal and maximal respiration, ATP production, spare respiratory capacity, proton leak and non-mitochondrial respiration in real-time in a minimal amount of macrophages.
Therefore, we modified and improved the manufacturer&#8217;s protocols as follows. The standard Mito Stress Test (# 103015-100) suggests using medium with glucose and pyruvate and a protocol with 3 injections (OM, FCCP, AA/Rot). We opt to start the assay with glucose/pyruvate-free medium, add glucose as a first injection in port A (like in the Glycolysis Stress Test # 103020-100) and add pyruvate together with the FCCP uncoupler in port C. In this way, all relevant glycolysis parameters are derived from the ECAR measurements 1-9 and all relevant information about mitochondrial function from OCR measurements 4-15.
Note that it is critical to inject pyruvate together with FCCP to fuel maximal respiration upon uncoupling. Before using this combined protocol, one should also verify that 2-Deoxy-D-Glucose (2-DG) lowers ECAR levels after the assay to the basal values (1-3) and that the recorded ECAR rates 4-6 are indeed due to glycolysis. Additionally, one should comprehend that the compound concentrations and cell numbers used in this manuscript are valid for bone marrow-derived macrophages and Raw264.7 macrophage cell lines and should be optimized for other cell types. Moreover, one should note that M-CSF which is used to generate bone marrow-derived macrophages promotes an M2-like anti-inflammatory phenotype. The presented results from bone marrow-derived macrophages may therefore be not fully comparable to measurements with primary tissue resident macrophages or macrophages cell lines that do not require M-CSF during cell culturing.
Compared to existing methods, this protocol requires minimal amounts of macrophages and quickly provides virtually all fundamental metabolic cell characteristics. This results in enough surplus cells to perform other immunological assays and even the cells used for bioenergetics profiling could still be used for other purposed after the assay. Moreover, the particular 96 well plate format allows assessing multiple conditions in real time at the same time. Yet, the variation between individual wells can be considerable and therefore the use of at least 4 wells per condition is often desired. Taken this limitation into account, the described extracellular flux analysis still allows to run more than 10 experimental conditions (n = 6) at once, which is much more than traditional techniques.
Overall, this article provides an easy and reproducible functional assay that allows measuring all relevant glycolysis and mitochondrial parameters in polarized macrophage subsets. This technique can serve as a future application to assess macrophage function and to evaluate the effect of distinct manipulations (e.g. gene knock down, new pharmaceuticals, etc.) on the macrophage&#8217;s (metabolic) phenotype. As such, extracellular flux data can be used in conjunction with other assays to allow in-depth characterisation of the macrophage activation status.

While macrophages play a central role in virtually all diseases, the molecular mechanisms that regulate their phenotype are still not fully unraveled. Macrophages display high heterogeneity and in response to the microenvironment adopt different phenotypes1. LPS(+IFN&#947;)-induced classically activated M1 or M(LPS) macrophages promote inflammation and protect the host against different types of microbial threats2. Others use IFN&#947; alone or in combination with TNF as stimuli to elicit M1 polarization. IL-4 and/or IL-13 induces alternative macrophage activation and these M2 or M(IL-4) cells are potent suppressors and controllers of ongoing immune responses3. Polarized macrophages display a distinct regulation of cellular metabolism with LPS-activated M1 macrophages undergoing a metabolic switch to enhanced glycolysis4-6. Conversely, enhanced fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) provides sustained energy in IL-4-induced M2 macrophages7-9. Thus, altered metabolism is not only a characteristic of polarized macrophage subsets, it is also a prerequisite for proper polarization and inflammatory regulation. Importantly, the inhibition of glycolysis or OXPHOS/FAO has been demonstrated to impair M1 or M2 activation, respectively8,10. As such, identifying metabolic changes in macrophages could be applied as a tool to assess their polarization status and inflammatory potential.
A consistent assay that measures macrophage metabolism could therefore be used to predict whether a drug, gene knock down or other treatment affects macrophage polarization and function. In this video, an extracellular flux analyzer is used to characterize the bioenergetics profiles of na&#239;ve, M1 and M2 macrophages.
This manuscript details an optimized protocol that allows the measurement of all relevant glycolysis parameters (glycolysis, maximal glycolysis and glycolytic reserve) and mitochondrial function characteristics (total respiration, basal mitochondrial respiration, ATP production, proton leak, maximal respiration and spare respiratory capacity) in one single assay. Using this experimental setup, bioenergetics can be compared between control and &#8216;altered&#8217; (e.g. gene knock down, transgenic overexpression or pharmacological treatment) cells.

<table><tbody><tr><td>23G and 25G needles</td><td>Becton Dickinson</td><td>#300800 and #300600</td><td>To flushed bone marrow</td></tr><tr><td>10 ml syringes</td><td>Becton Dickinson</td><td>#307736</td><td>To flushed bone marrow</td></tr><tr><td>Petri dishes</td><td>Greiner</td><td>#639161</td><td>To culture bone marrow cells</td></tr><tr><td>CyQUANT Cell Proliferation Assay kit&amp;nbsp;</td><td>Molecular Probes</td><td>#C7026</td><td>For cell quantification at a later time point (works only for adherent cells)</td></tr><tr><td>CyQUANT Direct Cell Proliferation Assay kit&amp;nbsp;</td><td>Molecular Probes</td><td>#35011</td><td>For cell quantification immediately after assay (non-adherent cells)</td></tr><tr><td>XF&lt;sup&gt;e&lt;/sup&gt;96 cell culture microplate</td><td>Seahorse Bioscience</td><td>#101085-004</td><td>96 well plate in which cells are cultured and in which assay is done</td></tr><tr><td>recombinant mouse interleukin-4 (IL-4)</td><td>Peprotech</td><td>#214-14-B</td><td>Used to induced alternative macrophage activation</td></tr><tr><td>lipopolysaccharide (LPS)</td><td>Sigma</td><td>#L2637</td><td>Pro-inflammatory stimulus</td></tr><tr><td>Seahorse Sensor Cartridge&amp;nbsp;</td><td>Seahorse Bioscience</td><td>#102416-100</td><td>Contains the probes for pH and O2 measurement</td></tr><tr><td>Seahorse XF Calibrant&amp;nbsp;</td><td>Seahorse Bioscience</td><td>#100840-000</td><td>Needed to hydrate the probes overnight&amp;nbsp;</td></tr><tr><td>XF base medium&amp;nbsp;</td><td>Seahorse Bioscience</td><td>#102353-100</td><td>Buffer-free medium that allows efficient measurement of pH changes</td></tr><tr><td>L-glutamine</td><td>Life Technologies</td><td>#25030-081</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>#G8769</td><td>Fuels glycolysis once added to the cells</td></tr><tr><td>oligomycin A</td><td>Sigma</td><td>#75351</td><td>Inhibits mitochondrial ATP synthase</td></tr><tr><td>FCCP( Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)</td><td>Sigma</td><td>#C2920</td><td>Uncouples mitochondrial respiration</td></tr><tr><td>100 mM Sodium Pyruvate solution</td><td>Lonza</td><td>#BE13-115E</td><td>Allows maximal mitochondrial respiration without the need for glycolysis</td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>#BE13-115EA8674</td><td>Inhibits complex III of the mitochondria</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>#BE13-115EA8674R8875</td><td>Inhibits complex I of the mitochondria</td></tr><tr><td>Casy Cell Counter</td><td>Roche Diagnostics</td><td>#05 651 697 001</td><td>Instrument to count cells</td></tr><tr><td>anti-CD16/CD32</td><td>eBioscience</td><td>#14-0161</td><td>Fc receptor block flow cytometry</td></tr><tr><td>anti-F4/80-APC-eFluor780</td><td>eBioscience</td><td>#47-4801</td><td>Antibody to stain macrophages</td></tr><tr><td>anti-CD11b-FITC</td><td>eBioscience</td><td>#11-0112</td><td>Antibody to stain macrophages</td></tr></tbody></table>

metabolic characterization of polarized m1 and m2 bone marrow-derived macrophages using real-time extracellular flux analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

After finishing the extracellular flux assay and cell quantification, data can be normalized for cell counts and analysed. This will typically yield ECAR and OCR plots as shown in Figure 1A and Figure 1B.
After the injection of glucose (A), the increase in ECAR represents the glycolysis rate. The additional increase in ECAR after ATP synthase inhibition with oligomycin (B) provides information about the glycolytic reserve and capacity (Figure 1A). When analysing the OCR values, oligomycin injection (B) allows calculation of the oxygen consumption used for mitochondrial ATP synthesis. FCCP (C) uncouples mitochondrial respiration and the corresponding OCR measurements yield data about the maximal and spare respiratory capacity. Finally, injection of rotenone (Rot) and antimycin A (AA) block mitochondrial complex I and III and the residual OCR represents the non-mitochondrial oxygen consumption (Figure 1B).
<img alt="Figure 1" src="/files/ftp_upload/53424/53424fig1.jpg" /> 
Figure 1: Metabolic parameters derived from a XF Extracellular flux assay. (A) Following parameters of cellular glycolysis are calculated from the ECAR values (in mpH/min): glycolysis, maximum glycolytic capacity and glycolytic reserve. (B) OCR measurements (in pMoles/min) are used to calculate the next fundamental parameters of mitochondrial function: basal respiration, ATP production, proton leak, maximum respiration and spare respiratory capacity. Gluc = glucose; OM = oligomycin A; FCCP = Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; AA = antimycin A; Rot = Rotenone <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53424/53424fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After the run, ECAR and OCR measurements 1 till 15 for each well can be exported to Excel and the following glycolytic parameters can be calculated from the ECAR measurements as follows:
Non-glycolytic acidification = avg. ECAR(1,2,3)
Glycolysis = avg. ECAR(4,5,6)&#8211; avg. ECAR(1,2,3)
Maximum glycolytic capacity = avg. ECAR(7,8,9)&#8211; avg. ECAR(1,2,3)
Glycolytic reserve= avg. ECAR(7,8,9) - avg. ECAR(4,5,6)
From the OCR rates, the next metabolic characteristics can be determined:
Non-mitochondrial respiration = avg. OCR(13,14,15)
Basal respiration = avg. OCR(4,5,6) - avg. OCR(13,14,15)
ATP production = avg. OCR(4,5,6) - avg. OCR(7,8,9)
Proton leak = avg. OCR(7,8,9) - avg. OCR(13,14,15)
Maximum respiration = avg. OCR(10,11,12) - avg. OCR(13,14,15)
Spare respiratory capacity (SRC) = avg. OCR(10,11,12) - avg. OCR(4,5,6)
<img alt="Figure 2" src="/files/ftp_upload/53424/53424fig2.jpg" /> 
Figure 2: Metabolic characteristics of na&#239;ve (M0), LPS- (M1) and IL-4- (M2) polarized macrophages. For each measurement, the mean and standard error of the mean (SEM) of 8 individual wells is presented. (A) Extracellular acidification rates (ECAR, in mpH/min) and (B) oxygen consumption rates (OCR, in pMoles/min) are measured for differentially polarized (M1 and M2) and na&#239;ve macrophages (M0). All measurements were normalized for cell count (as measured with CyQUANT) and the average cell count of the na&#239;ve macrophages was set at 1. Injection A = glucose; B = oligomycin; C = FCCP; D= antimycin A + Rotenone. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53424/53424fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As shown in Figure 2A, macrophage activation with LPS induces increased glycolytic metabolism. The differences between LPS and IL-4-treated macrophages are even more apparent when looking at oxygen consumption rates (OCR) in Figure 2B. Indeed, while the maximal oxidative metabolism is highly suppressed in M(LPS), IL-4 induces basal and especially maximal respiration in M2 macrophages. One should note that increased glycolytic is rather unique to M(LPS) macrophages and is not necessarily a characteristic of M1 macrophages since M(IFN&#947;) macrophages do not display enhance glycolysis.
Overall, this extracellular flux analysis demonstrates that IL-4-induced M2 macrophages are characterized by enhanced oxidative metabolism and especially a high spare respiratory capacity (SRC), whereas LPS-activated macrophages display enhanced glycolytic metabolism and do not possess any spare respiratory capacity.

Watch this Scientific Journal Video about Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis at JoVE.com

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

35.4K Views.  Academic Medical Center, Amsterdam. Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage’s metabolism and phenotype.

Video: Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

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

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Biochemistry

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-&#947; and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4- or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong up-regulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1-polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected and Mtb-infected cells at 24-hours post-infection. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs in different areas of research.

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

This protocol provides a method to study Mycobacterium tuberculosis infection in human M1- or M2-polarized macrophages based on differentiation of peripheral-blood-monocytes to macrophage-like cells that are infected with the GFP-labeled virulent strain H37Rv, and analyzed with flow cytometry using a 10-color panel including expression of selected M1/M2 markers.

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Assessing Immunomodulatory Effects of Implant Surfaces on Macrophages

Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces 

Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the first line of immune system interaction with implant surfaces, play a bidirectional role in modulating the inflammation-regeneration balance. For a deep understanding and the evaluation of the reactions between implant materials and immune responses, reliable in vitro methods and protocols are pivotal. Among different in vitro models, primary monocyte-derived macrophages (MDMs) present an excellent model for investigating macrophage-implant interactions. We have implemented an experimental protocol to evaluate the polarization of MDMs into M1 (classically activated) and M2 (alternatively activated) macrophages on implant surfaces. We isolated blood monocytes from healthy donors and differentiated them into macrophages using macrophage colony-stimulating factor (M-CSF). Differentiated macrophages were cultured on implant surfaces and polarized into M1 and M2 subtypes. M1 polarization was achieved in the presence of interferon (IFN)-&#947; and lipopolysaccharide (LPS), while M2 polarization was performed in a medium containing interleukin (IL)-4 and IL-13. We evaluated macrophage phenotypes by Enzyme-linked&#160;Immunosorbent Assay (ELISA), confocal laser scanning microscopy (CLSM), and quantitative real-time PCR (qRT-PCR) based on panels of secreted cytokines, cell surface markers, and expressed genes. The extracted RNA was transformed into complementary DNA (cDNA), and qRT-PCR was used to quantify mRNA related to M1 and M2 macrophages. Accordingly, M1 macrophages have been characterized by higher expression of proinflammatory Tumor necrosis factor (TNF-&#945;) cytokine and CCR7 surface marker compared to M2&#160;macrophages, which exhibited higher levels of CD209 and CCL13. Consequently, CCR7 and CD209 were identified as specific and reliable markers of M1 and M2 macrophage subtypes by immunostaining and visualizing by CLSM. Further confirmation was achieved by ELISA detecting elevated TNF-&#593; level in M1 and increased CCL13 in M2 cells. The proposed markers and experimental setup can be used effectively to assess the immunomodulatory potential of implants.

Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces

Here, we present a detailed protocol for assessing the immunomodulatory potential of implant surfaces in vitro, aiming to improve the reliability and reproducibility of current protocols and promoting further research. Secretory cytokine profiles, mRNA expression, and cell surface markers were monitored using blood monocyte-derived macrophages to investigate macrophage polarization cultivated on titanium.

Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the ...

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

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