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. 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=Targeting+macrophage+Histone+deacetylase+3+stabilizes+atherosclerotic+lesions.">Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions.</a> EMBO Mol Med. 6, 1124-1132 (2014).</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=Alternatively+activated+macrophages+engage+in+homotypic+and+heterotypic+interactions+through+IL+4+and+polyamine+induced+E+cadherin+catenin+complexes.">Alternatively activated macrophages engage in homotypic and heterotypic interactions through IL 4 and polyamine induced E cadherin catenin complexes.</a> Blood. 114, 4664-4674 (2009).</li><li>Van den 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=Inhibiting+epigenetic+enzymes+to+improve+atherogenic+macrophage+functions.">Inhibiting epigenetic enzymes to improve atherogenic macrophage functions.</a> Biochem Biophys Res Commun. 455, 396-402 (2014).</li><li>Galvan Pena, S., O Neill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+reprograming+in+macrophage+polarization.">Metabolic reprograming in macrophage polarization.</a> Frontiers in immunology. 5, 420 (2014).</li><li>Zhu, L., Zhao, Q., Yang, T., Ding, W., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+metabolism+and+macrophage+functional+polarization.">Cellular metabolism and macrophage functional polarization.</a> International reviews of immunology. 34, 82-100 (2015).</li></ol>

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 border="0">
	<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&#160;</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&#160;</td>
			<td>Molecular Probes</td>
			<td>#35011</td>
			<td>For cell quantification immediately after assay (non-adherent cells)</td>
		</tr>
		<tr>
			<td>XFe96 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&#160;</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&#160;</td>
			<td>Seahorse Bioscience</td>
			<td>#100840-000</td>
			<td>Needed to hydrate the probes overnight&#160;</td>
		</tr>
		<tr>
			<td>XF base medium&#160;</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 ...

metabolic-characterization-polarized-m1-m2-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

35.2K 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 ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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 ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

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 ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...