AM is funded by the Young Group Leader Program (ATIP/Avenir Inserm-CNRS), by la Ligue Nationale contre le Cancer and la Fondation ARC pour la recherche sur le Cancer. We thank Mariette Matondo from the Mass Spectrometry for Biology platform (UTECHS MSBIO, Pasteur Institute, Paris). We thank Lauren Anderson for her reading of the manuscript.

Human blood samples (LRSC) from healthy, de-identified donors were obtained from EFS (French National Blood Service) as part of an authorized protocol (CODECOH DC-2018&#8211;3114). Donors gave signed consent for the use of blood.
1. Media and Buffer Preparation
<ol>
	<li>Prepare the macrophage medium [RPMI glutamax + 10 mM HEPES + 1x non-essential amino acids (NEAA)] and warm it to 37 &#176;C.</li>
	<li>Prepare the macrophage medium + 10% human serum from AB plasma (SAB), filter it (0.22 &#181;m filter), then warm it to 37 &#176;C (referred to as macrophage medium + 10% SAB hereafter).</li>
	<li>Prepare the sorting buffer [1x phosphate buffered saline (PBS) + 0.5% bovine serum albumin (BSA) + 2 mM ethylenediaminetetraacetic acid (EDTA)], filter it (0.22 &#181;m filter), and maintain it at 4 &#176;C.</li>
</ol>
2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Leukoreduction System Chamber (LRSC)
<ol>
	<li>Put 15 mL of density gradient cell separation solution (see Table of Materials) in a 50 mL centrifugation tube so it can warm to room temperature (RT) before receiving the LRSC. 
	NOTE: Density depends on temperature. As this product is stored at 4 &#176;C, this step must be done in advance so it can equilibrate to RT.</li>
	<li>Empty the LRSC into a 50 mL centrifugation tube, add up to 50 mL of 1x PBS, and mix. Very slowly, add 25 mL of the mix prepared during step 2.2 on top of 15 mL of density gradient solution warmed up during step 2.1. 
	NOTE: Be careful not to mix the phases during this step. The blood must be added on the density gradient solution without any disturbance of this phase.</li>
	<li>Centrifuge both centrifugation tubes for 25 min at 700 x g without breaks. 
	NOTE: At the end of the density gradient centrifugation, the layers from bottom to top are: the erythrocytes and granulocytes forming the pellet, density gradient solution phase, layer of PBMCs, and plasma.</li>
	<li>With a pipet, pass through the plasma phase without aspirating it and collect the PBMC layer into a new 50 mL centrifugation tube. Add up to 50 mL of 1x PBS to the PBMCs as a washing step and centrifuge for 10 min at 300 x g.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 40 mL of macrophage medium.</li>
</ol>
3. Magnetic Labeling and Isolation of CD14+ Cells (Monocytes)
<ol>
	<li>Count the PBMCs in a Malassez chamber. Withdraw the amount of PBMCs necessary for conducting the experiment (typically 100 to 300 x 106 cells), place them in a centrifugation tube, and centrifuge for 10 min at 300 x g.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 80 &#181;L of the sorting buffer prepared during step 1.3 per 107 PBMCs. Add 20 &#181;L of CD14 microbeads per 107 PBMCs. Mix well and incubate for 15 min at 4 &#176;C under constant agitation.</li>
	<li>Add 1 mL of sorting buffer per 107 PBMCs as a washing step and centrifuge for 10 min at 300 x g. Aspirate the supernatant and resuspend the pellet in 500 &#181;L of sorting buffer per 108 PBMCs.</li>
	<li>Place a column in the magnetic field of the separator. Prepare the column by rinsing it with 3 mL of sorting buffer.</li>
	<li>Apply the cell suspension onto the column. The column depends on the number of cells to be isolated (here, LS columns for up to 109 PBMCs are used). Collect flow-through containing unlabeled cells. 
	NOTE: Starting at this step, all the tubes (negative and positive selections) are kept for later checking of the different steps by flow cytometry.</li>
	<li>Wash the column with 3 x 3 mL of sorting buffer. Collect unlabeled cells passing through the same tube from step 3.12. Perform washing steps by adding sorting buffer, be careful not to dry your column. Place a collection tube under the column and remove it from the separator.</li>
	<li>Pipette 5 mL of sorting buffer into the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. To increase purity of the CD14+ cells, the eluted fraction is enriched over a second column.</li>
	<li>Repeat steps 3.4 to 3.7 with a new column.</li>
</ol>
4. Plating of Monocytes
<ol>
	<li>Count the monocytes in a Malassez chamber. Check the purity of the CD14+ cells by flow cytometry. Withdraw the amount of monocytes necessary for the experiment and place them in a centrifugation tube.</li>
	<li>Centrifuge for 10 min at 300 x g. Aspirate the supernatant and resuspend the monocyte pellet in macrophage medium. Plate the cells and let them settle for 50 min to 1 h. Aspirate the medium and replace it with macrophage medium + 10% SAB + 25 ng/mL macrophage colony stimulating factor (M-CSF) to induce differentiation.</li>
</ol>
5. Polarization of Macrophages at Day 6
<ol>
	<li>Aspirate the medium. Replace it with macrophage medium + 10% SAB with various stimuli. For example, add 10 ng/mL interferon gamma (INF&#947;) + 1 ng/mL lipopolysaccharide (LPS) to obtain M1 polarization, or 20 ng/mL interleukin 4 (IL4) + 20 ng/mL interleukin 13 (IL13) for M2 polarization. 
	NOTE: The stimulation can be performed between 24 and 48 h before proceeding to other tests.</li>
	<li>Harvest cells using a detaching solution or a cell scraper.</li>
</ol>
6. Cell Culture Under Low Oxygen Conditions
<ol>
	<li>Starting from step 4, maintain the monocytes and then macrophages in an oxygen-controlled environment to perform hypoxic condition analysis. Use a hypoxia working station in order to maintain cells under the desired oxygen partial pressure during the experiment. 
	NOTE: When working under low oxygen pressure, it is important to prepare all media and washing buffers under the station and wait sufficiently to obtain the correct partial pressure in the liquid. For example, 10 mL of PBS in a 60 mm Petri dish requires roughly 2 h to reach 25 mmHg for O2 partial pressure starting from atmospheric pressures (as we have measured it using a fiber-optic oxygen sensor). In many hypoxic stations or incubators, the oxygen pressure is set as a percentage of the atmospheric pressure. If precise measurements are necessary, it is better to use a station authorizing to directly set the oxygen pressure in mmHg.</li>
</ol>
7. Lysis and In-Gel Digestion (Protocol 1)
NOTE: In this and the following sections, two protocols used to obtain peptides and perform LC-MS/MS analysis are described. Protocol 1 describes cell lysis and in-gel fractionation and digestion, and protocol 2 describes in-solution cell lysis followed by in-solution digestion and fractionation using an isoelectric focusing method.
<ol>
	<li>Perform cell lysis in Laemmli buffer [234 mM Tris-HCL (pH 6.8), 7.5% SDS, 37% glycerol, 33.3% (v/v) &#946;-mercaptoethanol, bromophenol blue 0.2% w/v]. Load the protein equivalent of 300,000 cells for each sample on 4-12% bis-Tris acrylamide gels.</li>
	<li>Control the duration of the electrophoretic migration to allow each protein sample to be split into 6 gel bands as exemplified in Figure 3.</li>
	<li>Fix the gel with a fixing solution (30% ethanol + 7.5% acetic acid for 20 min), then add the staining solution (R-250 Coomassie blue for 45 min). Add the destaining solution (30% ethanol + 7.5% acetic until bands appear) before excising the protein bands with a clean scalpel.</li>
	<li>Dice each excised band before introduction in 500 &#181;L tubes. A clean glass surface is warranted to avoid contamination with keratins (5% SDS solution in deionized water can be used to clean surfaces).</li>
	<li>Wash the gel slices 3 times in 200 &#181;L of 25 mM ammonium bicarbonate for 20 min at 37 &#176;C, followed by one wash in 25 mM ammonium bicarbonate and acetonitrile (50% v/v). Dehydrate the gel pieces with 200 &#181;L of 100% acetonitrile for 10 min.</li>
	<li>Incubate each gel piece with 10 mM DTT (dithiothreitol) in 25 mM ammonium bicarbonate for 45 min at 56 &#176;C (200 &#181;L), followed by 55 mM iodoacetamide in 25 mM ammonium bicarbonate (200 &#181;L) for 35 min in the dark at RT.</li>
	<li>To stop alkylation, incubate each gel piece with 200 &#181;L of 10 mM DTT in 25 mM ammonium bicarbonate for 10 min at RT. Wash the gel pieces in 200 &#181;L of 25 mM ammonium bicarbonate, then dehydrate with 200 &#181;L of 100% acetonitrile for 10 min.</li>
	<li>Digest the proteins overnight at 37 &#176;C with Trypsin/Lys-C mix according to the manufacturer&#8217;s instructions.</li>
	<li>Extract the resulting peptides from gel pieces by adding 50 &#956;L of 50% acetonitrile for 15 min, then 50 &#956;L of 5% formic acid for 15 min, and finally, 50 &#956;L of 100% acetonitrile for 15 min. Pool and dry each fraction in low-absorption tubes to limit adsorption of peptides and sample loss. Store the samples at -80 &#176;C until further analysis.</li>
</ol>
8. Protein Extraction and In-Solution Digestion (Protocol 2)
<ol>
	<li>Perform cell lysis (2 x 106 cells) with 150 &#181;L of the following lysis buffer:
	<ol>
		<li>7 M urea, 2 M thiourea, 40 mM Tris, and 4% CHAPS, supplemented with protease inhibitors (complete mini, EDTA-free protease inhibitor cocktail).</li>
	</ol>
	</li>
	<li>Homogenize the solutions for 30 min at RT with a thermoshaker. Centrifuge at 13,800 x g for 20 min RT and keep the supernatant.</li>
	<li>Remove contaminants with a 2D clean-up kit:
	<ol>
		<li>The kit contains precipitant solution, co-precipitant solution, wash buffer, and wash additive.</li>
		<li>Add 300 &#181;L of precipitant solution and mix well. Incubate on ice for 15 min. Add 300 &#181;L of co-precipitant solution. Centrifuge the tubes (at least) at 12,000 x g for 5 min. A small pellet should be visible. Proceed rapidly to the next step to avoid resuspension or dispersion of the pellet. Remove the supernatant without disturbing the pellet.</li>
		<li>Centrifuge the tubes again with the cap-hinge and pellet facing outward to bring any remaining liquid to the bottom of the tube. A brief pulse is sufficient. There should be no visible liquid remaining in the tubes.</li>
		<li>Without disturbing the pellet, add 40 &#181;L of co-precipitant solution. Let the tube sit on ice for 5 min. Centrifuge for 5 min, then remove and discard the wash. Add 25 &#181;L of de-ionized water. Vortex each tube for 5-10 s. The pellet should disperse but not dissolve in the water.</li>
		<li>Add 1 mL of wash buffer (pre-chilled for at least 1 h at -20&#176;C) and 5 &#181;L of wash additive. Vortex until the pellet is fully dispersed. Incubate the tubes at -20&#176;C for at least 30 min. Vortex for 20-30 s every 10 min 
		NOTE: The tubes can be stored at -20&#176;C for up to 1 week with minimal protein degradation or modification.</li>
		<li>Centrifuge the tubes (at least) at 12,000 &#215; g for 5 min. Carefully remove and discard the supernatant. A white pellet should be visible. Allow the pellet to airdry for no more than 5 min (if the pellet is too dry, it will be difficult to resuspend).</li>
	</ol>
	</li>
	<li>Resuspend the protein pellet in 300 &#181;L of 8 M urea and 0.1 M ammonium bicarbonate. Vortex strongly for 1 min. Determine the protein concentration using a colorimetric assay.</li>
</ol>
9. In-Solution Digestion (Protocol 2)
<ol>
	<li>Reduce disulfide bridges by adding 5.1 &#181;L of a 700 mM DTT aqueous solution (final concentration 12.5 mM) to the resuspended proteins from step 8.4 and incubate at 37 &#176;C for 30 min with a thermoshaker. Alkylate cysteine residues by adding 20.3 &#181;L of a 700 mM iodoacetamide aqueous solution (final concentration 40 mM) and incubating at 25&#176;C for 30 min in the dark with a thermoshaker.</li>
	<li>Add 990 &#181;L of 0.1 M ammonium bicarbonate to the sample. Add a corresponding volume of Trypsin/Lys-C mix (enzyme:substrate ratio 1:100 w/w). Incubate at 37&#176;C overnight with a thermoshaker.</li>
</ol>
10. Clean-up Cartridge (Protocol 2)
<ol>
	<li>Wet a cartridge with 1 column-volume (1 mL) of methanol. Clean the cartridge with 1 column-volume (1 mL) of 80% acetonitrile/HPLC-grade water and discard the flow-through. Equilibrate the cartridge with 4 column-volumes (4 mL) of 0.1% formic acid/HPLC-grade water and discard the flow-through.</li>
	<li>Acidify samples with 90 &#181;L of 10% formic acid or water to pH 2-3 (check the pH with a pH indicator). Load the acidified samples and collect the flow-through. Reload the flow-through (containing the not-retained peptides). Wash the cartridge with 6 column-volume (6 mL) of 0.1% formic acid/HPLC-grade water.</li>
	<li>Elute peptides from the cartridge with 1 column-volumes (1 mL) of 0.1% formic acid/50% acetonitrile/HPLC-grade water. Transfer to a 1.5 mL microcentrifuge tube. Concentrate the sample using a vacuum concentrator (150 x g, vacuum at 160 mBar).</li>
</ol>
11. Fractionation by Isoelectric Focusing (Protocol 2)
NOTE: Peptides are separated according to their isoelectric points using an off-gel fractionator on a 13 cm strip covering a pH range from 3 to 10. We used the following protocol provided by the supplier (summarized below):
<ol>
	<li>Prepare the following solutions: solution A (600 &#181;L of glycerol solution, 60 &#181;L of OFFGEL buffer, 4.34 mL of ultrapure water) and solution B (1.776 mL of solution A and 444 &#181;L of ultrapure water).</li>
	<li>Assemble the IPG strips, frames, and electrodes according to the manufacturer&#8217;s instructions.</li>
	<li>Resuspend the sample with 1.8 mL of solution B. Add 40 &#181;L of solution B into each well. Load 150 &#181;L of sample into each well.</li>
	<li>Select the default method for peptides: OG12PE00 (OFFGEL default method for peptides for use with a 3100 OFFGEL Low Res Kit, pH 3-10, 12-well frames. Wait until this method has been completed (~20 h). Collect the fractions in properly labeled tubes.</li>
</ol>
12. Clean-up Harvard Apparatus Column Reverse C18 Post-IEF (Protocol 2)
<ol>
	<li>Progressively add a few &#956;L at a time of 1% TFA in de-ionized water to each fraction to acidify the sample. Check using pH paper that the pH is about 3 or below.</li>
	<li>Prepare the following solutions: solution 1 (5 mL of acetonitrile, 10 &#181;L of formic acid, 4.99 mL of ultrapure water) and solution 2 (0.5 mL of acetonitrile, 10 &#181;L of formic acid, 9.49 mL of ultrapure water).</li>
	<li>Pre-wet the spin column with 150 &#181;L of solution 1. Centrifuge for 90 s at 750 x g and discard the flow-through. Wash the spin column with 150 &#956;L of solution 2. Centrifuge for 90 s at 750 x g and discard the flow-through.</li>
	<li>Pass the fraction through the column. Centrifuge for 90 s at 750 x g and discard the flow-through. Wash with 150 &#956;L of solution 2. Centrifuge for 90 s at 750 x g and discard the flow-through.</li>
	<li>Elute the fraction with 50 &#956;L of solution 1. Centrifuge for 90 s at 750 x g. Repeat these steps once more.</li>
	<li>Dry-fraction using a vacuum concentrator (150 x g, vacuum 160 mBar) and store at -80&#176;C</li>
</ol>
13. Analysis of Proteomic Data and Bioinformatics18
<ol>
	<li>Analyze data obtained by a nano-LC MS/MS mass spectrometer using quantification software such as MaxQuant (version 1.5.2.8) and the Andromeda search engine.</li>
	<li>Set false discovery rate (FDR) to 1% for both proteins and peptides and a minimum length of 7 amino acids. Set enzyme specificity as C-terminal to Arg and Lys. Allow 2 missed cleavages at proline bonds. Select carbamidomethylation of cysteine as a fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications.</li>
	<li>Further analyze the data with statistical analysis software. Perform a functional enrichment analysis using FunRich software (www.funrich.org/). Perform a gene ontology enrichment analysis using DAVID software (https://david.ncifcrf.gov/).</li>
</ol>

<ol>
	<li>Okabe, Y., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+biology+perspective+on+macrophages.">Tissue biology perspective on macrophages.</a> Nature Immunology. 17 (1), 9-17 (2016).</li><li>Sica, A., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+plasticity+and+polarization:+in+vivo+veritas.">Macrophage plasticity and polarization: in vivo veritas.</a> The Journal of Clinical Investigation. 122 (3), 787-795 (2012).</li><li>Murray, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Polarization.">Macrophage Polarization.</a> Annual Review of Physiology. 79, 541-566 (2017).</li><li>Chinetti-Gbaguidi, G., Staels, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+metabolic+disorders:+functions+and+regulation.">Macrophage polarization in metabolic disorders: functions and regulation.</a> Current Opinion in Lipidology. 22 (5), 365-372 (2011).</li><li>Chow, A., Brown, B. D., Merad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+mononuclear+phagocyte+system+in+the+molecular+age.">Studying the mononuclear phagocyte system in the molecular age.</a> Nature Reviews Immunology. 11 (11), 788-798 (2011).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nature Reviews Immunology. 8 (12), 958-969 (2008).</li><li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nature Reviews Immunology. 11 (11), 723-737 (2011).</li><li>Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J., Biswas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+multidimensional+concept+of+macrophage+ontogeny,+activation+and+function.">New insights into the multidimensional concept of macrophage ontogeny, activation and function.</a> Nature Immunology. 17 (1), 34-40 (2016).</li><li>Xue, 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=Transcriptome-based+network+analysis+reveals+a+spectrum+model+of+human+macrophage+activation.">Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.</a> Immunity. 40 (2), 274-288 (2014).</li><li>Csete, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+in+the+cultivation+of+stem+cells.">Oxygen in the cultivation of stem cells.</a> Annals of the New York Academy of Sciences. 1049, 1-8 (2005).</li><li>Johansson, 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=Functional,+morphological,+and+phenotypical+differences+between+rat+alveolar+and+interstitial+macrophages.">Functional, morphological, and phenotypical differences between rat alveolar and interstitial macrophages.</a> American Journal of Respiratory Cell and Molecular Biology. 16 (5), 582-588 (1997).</li><li>Pfau, J. C., Schneider, J. C., Archer, A. J., Sentissi, J., Leyva, F. J., Cramton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+oxygen+tension+affects+phenotype+in+cultured+bone+marrow-derived+macrophages.">Environmental oxygen tension affects phenotype in cultured bone marrow-derived macrophages.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 286 (2), L354-L362 (2004).</li><li>Grodzki, A. C. G., Giulivi, C., Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+modulates+differentiation+and+primary+macrophage+functions+in+the+human+monocytic+THP-1+cell+line.">Oxygen tension modulates differentiation and primary macrophage functions in the human monocytic THP-1 cell line.</a> PLoS One. 8 (1), e54926 (2013).</li><li>Court, M., Petre, G., Atifi, M. E., Millet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Signature+Reveals+Modulation+of+Human+Macrophage+Polarization+and+Functions+Under+Differing+Environmental+Oxygen+Conditions.">Proteomic Signature Reveals Modulation of Human Macrophage Polarization and Functions Under Differing Environmental Oxygen Conditions.</a> Molecular &amp; Cellular Proteomics. 16 (12), 2153-2168 (2017).</li><li>Cox, 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=Accurate+proteome-wide+label-free+quantification+by+delayed+normalization+and+maximal+peptide+ratio+extraction,+termed+MaxLFQ.">Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.</a> Molecular &amp; Cellular Proteomics. 13 (9), 2513-2526 (2014).</li><li>Cox, J., Neuhauser, N., Michalski, A., Scheltema, R. A., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Andromeda:+a+peptide+search+engine+integrated+into+the+MaxQuant+environment.">Andromeda: a peptide search engine integrated into the MaxQuant environment.</a> Journal of Proteome Research. 10 (4), 1794-1805 (2011).</li><li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes.">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nature Protocols. 1 (6), 2856-2860 (2006).</li><li>Court, 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=Toward+a+standardized+urine+proteome+analysis+methodology.">Toward a standardized urine proteome analysis methodology.</a> Proteomics. 11 (6), 1160-1171 (2011).</li><li>Wi&#347;niewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nature Methods. 6 (5), 359-362 (2009).</li><li>Liebler, D. C., Ham, A. -. J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+filter-based+sample+preparation+for+shotgun+proteomics.">Spin filter-based sample preparation for shotgun proteomics.</a> Nature Methods. 6 (11), 785-786 (2009).</li><li>Hubner, N. C., Ren, S., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+separation+with+immobilized+pI+strips+is+an+attractive+alternative+to+in-gel+protein+digestion+for+proteome+analysis.">Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis.</a> Proteomics. 8 (23-24), 4862-4872 (2008).</li><li>Park, 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=Informed-Proteomics:+open-source+software+package+for+top-down+proteomics.">Informed-Proteomics: open-source software package for top-down proteomics.</a> Nature Methods. 14 (9), 909-914 (2017).</li><li>Richter, A., Sanford, K. K., Evans, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+oxygen+and+culture+media+on+plating+efficiency+of+some+mammalian+tissue+cells.">Influence of oxygen and culture media on plating efficiency of some mammalian tissue cells.</a> Journal of the National Cancer Institute. 49 (6), 1705-1712 (1972).</li><li>Packer, L., Fuehr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+oxygen+concentration+extends+the+lifespan+of+cultured+human+diploid+cells.">Low oxygen concentration extends the lifespan of cultured human diploid cells.</a> Nature. 267 (5610), 423-425 (1977).</li><li>Lengner, C. 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=Derivation+of+pre-X+inactivation+human+embryonic+stem+cells+under+physiological+oxygen+concentrations.">Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations.</a> Cell. 141 (5), 872-883 (2010).</li><li>Sidoli, S., Kulej, K., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+proteomics+is+not+the+new+genomics+and+the+future+of+mass+spectrometry+in+cell+biology.">Why proteomics is not the new genomics and the future of mass spectrometry in cell biology.</a> Journal of Cell Biology. 216 (1), 21-24 (2017).</li></ol>

The authors declare no conflicts of interest.

Because proteomics is a powerful tool to study the expression of different proteins from a whole cell or subcellular compartments, optimization of the cell lysis protocol and digestion of proteins has been addressed by a number of studies. There are three main classes of methods, which include in-gel digestion (digestion of proteins in polyacrylamide gel matrix)17, digestion in solution18 and filter-aided sample preparation19. This last method, at fi...

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis, including removal of apoptotic cells and remodelling of the extracellular matrix1. These cells are characterized by a strong phenotypic plasticity2 that translates into a many possible activation states, which are also called polarizations. The precise molecular description of these various polarizations is a priority in the field of macrophage biology3. It has been proposed to classify these polarizations using the so-called M1/M2 dichotomy, in which M1 represents pro-inflammatory and M2 represents anti-inflammatory macrophages. This model fits well in various pathological situations like acute infections, allergy, and obesity4. However, in chronically inflamed tissues and cancer, it has been demonstrated that this classification is unable to grasp the broad phenotypic repertoire that macrophages present in certain cellular environments5,6,7. The current consensus is that macrophage polarization is better described using a multidimensional model to integrate specific microenvironmental signals8. This conclusion has been confirmed through transcriptomic analysis of human macrophages showing that the M1/M2 model is inefficient in describing the obtained polarizations9.
The study presented aims to provide a protocol to obtain proteomic signatures of various polarizations in human macrophages. We describe how to differentiate human macrophages in environments of various oxygen levels and obtain peptides from the whole macrophage proteome to perform a label-free quantification. This quantification allows the comparison of expression levels of various proteins. As research on stem cells has revealed the importance of oxygen as an environmental key parameter10, we seek to understand how this tissue parameter can influence macrophage polarization in humans. The partial pressure of oxygen has been found to range from 3 to 20% (of total atmospheric pressure) in the human body, where 20% corresponds roughly to what is commonly used in a cell culture incubator (the exact value is around 18.6% while taking the presence of water into account).
Previous work has shown that alveolar differ from interstitial macrophages from functional and morphological point of views11 and that these differences are probably partially due to the different oxygen levels to which they are exposed12. Furthermore, bone marrow-derived macrophages show an increased ability to phagocytize bacteria when exposed to a low oxygen environment12. The opposite effect has been found for THP1-differentiated human macrophages13, but these results support the idea that oxygen is a regulator of macrophage biology and that it is necessary to clarify this role at the molecular level in human macrophages. In a previous study, we have applied a proteomics approach to address these issues. By measuring expression levels for thousands of proteins simultaneously, we highlighted the impact of oxygen on polarization and provided a list of new molecular markers. We were also able to relate these findings to some macrophages functions. Notably, we found that the rate of phagocytosis of apoptotic cells was increased in IL4/IL13-polarized macrophages, which was linked to the upregulation of ALOX15 as revealed by the proteomic analysis14. In the present study, we describe how to perform such an analysis.

<table><tbody><tr><td>Hypoxia Working Station</td><td>Oxford Optronix</td><td>Hypoxylab</td><td></td></tr><tr><td>C6 Flow cytometer</td><td>BD</td><td>Accuri C6</td><td></td></tr><tr><td>Urea</td><td>Agilent Technologies</td><td>5188-6435</td><td></td></tr><tr><td>Formic acid (FA)</td><td>ARISTAR</td><td>450122M</td><td></td></tr><tr><td>R-250 Coomassie blue</td><td>Biorad</td><td>1,610,436</td><td></td></tr><tr><td>Lipopolysaccharide, E.Coli (LPS)</td><td>Calbiochem</td><td>437627</td><td></td></tr><tr><td>2D clean-up kit</td><td>GE Healthcare</td><td>80-6484-51</td><td></td></tr><tr><td>RPMI 1640 medium, glutamax supplement</td><td>Gibco</td><td>61870044</td><td></td></tr><tr><td>HEPES 1 M</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (NEAA) Solution 100X</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) 1X</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Harvard Apparatus column Reverse C18 micro spin column</td><td>Harvard Apparatus</td><td>74-4601</td><td></td></tr><tr><td>EDTA 0.5 M, pH 8.0</td><td>Invitrogen</td><td>AM9260G</td><td></td></tr><tr><td>NuPAGE Bis-Tris 4-12%</td><td>Life Technologies SAS</td><td>NP0321 BOX</td><td></td></tr><tr><td>CD14 Microbeads human</td><td>Miltenyi Biotec</td><td>130-050-201</td><td></td></tr><tr><td>MACS separation column LS</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>Macrophage colony-stimulating factor (M-CSF)</td><td>Miltenyi Biotec</td><td>130-096-485</td><td></td></tr><tr><td>Interleukin 4 (IL4)</td><td>Miltenyi Biotec</td><td>130-093-917</td><td></td></tr><tr><td>Interleukin 13 (IL13)</td><td>Miltenyi Biotec</td><td>130-112-410</td><td></td></tr><tr><td>Interferon gamma (INF&amp;gamma;)</td><td>Miltenyi Biotec</td><td>130-096-482</td><td></td></tr><tr><td>CD14-FITC (clone T&amp;Uuml;K4)</td><td>Miltenyi Biotec</td><td>130-080-701</td><td></td></tr><tr><td>MACSmix Tube Rotator</td><td>Miltenyi Biotec</td><td>130-090-753</td><td></td></tr><tr><td>Trifluoroacetic Acid (TFA)</td><td>Pierce</td><td>28904</td><td></td></tr><tr><td>Trypsin/Lys-C Mix</td><td>PROMEGA</td><td>V5073</td><td></td></tr><tr><td>Complete Mini, EDTA-free Protease Inhibitor cocktail</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>Density Gradient Solution (Histopaque 1077)</td><td>Sigma Aldrich</td><td>10771-100ML</td><td></td></tr><tr><td>Accumax</td><td>Sigma Aldrich</td><td>A7089-100ML</td><td></td></tr><tr><td>Human Serum from human male AB plasma (SAB)</td><td>Sigma Aldrich</td><td>H4522-100ML</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) solution 30%</td><td>Sigma Aldrich</td><td>A9576-50ML</td><td></td></tr><tr><td>Trisma-base</td><td>Sigma Aldrich</td><td>T1503</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>49767</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M3148</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma Aldrich</td><td>114405</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS) 20%</td><td>Sigma Aldrich</td><td>5030</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma Aldrich</td><td>9830</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>34888</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma Aldrich</td><td>43819</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma Aldrich</td><td>57670</td><td></td></tr><tr><td>Thiourea</td><td>Sigma Aldrich</td><td>T8656</td><td></td></tr><tr><td>CHAPS</td><td>Sigma Aldrich</td><td>C9426</td><td></td></tr><tr><td>Micro BCA Assay Kit</td><td>ThermoFisher</td><td>23235</td><td></td></tr><tr><td>5 mL sterile plastic pipette</td><td>VWR</td><td>612-1685</td><td></td></tr><tr><td>Thermomixer C Eppendorf</td><td>VWR</td><td>460-0223</td><td></td></tr><tr><td>Sep-Pak tC18 reverse phase cartridges, 100 mg</td><td>Waters</td><td>WAT036820</td><td></td></tr></tbody></table>

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.

Starting from peripheral blood mononuclear cells (PBMCs) obtained by differential centrifugation, the protocol permits the obtaining of a population of CD14+ monocytes with an assessed purity of more than 98% by flow cytometry (Figure 1). These monocytes are secondarily differentiated toward various polarizations (Figure 2). When a fractionation on gel is chosen, the migration on SDS-page gels is adapted to obtain the ...

Watch this Scientific Journal Video about Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment at JoVE.com

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

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. ...

proteomic-analysis-human-macrophage-polarization-under-low-oxygen

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

7.9K Views.  Team Mechanobiology, Immunity and Cancer, Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309. 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.

Video: Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

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 ...

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

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.

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 ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Neuroscience

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. ...

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

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.

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.

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Proteomic Profiling of Macrophages by 2D Electrophoresis

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

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

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

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

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

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