This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

The protocol follows the guidelines of our institutions human research ethics committee. Buffy coat from healthy human donors were obtained from the Regional Blood Transfusion Center (Lille, France). Samples obtained from the buffy coats are declared as an Inserm collection (n&#176;DC2010-1209).
1. Material and Culture Media Preparation
<ol>
	<li>Dilute 10x Phosphate Buffer Saline (PBS) in sterile distilled water to obtain 1x PBS.</li>
	<li>Make RPMI 1640 medium supplemented with gentamicin (40 &#956;g/ml) and L-glutamine (2 mM), with and without 10% pooled human serum.</li>
</ol>
2. Primary Cultures of Monocyte-derived Macrophages (MDM)
<ol>
	<li>Dilute buffycoat (25 ml) with 1x PBS. Carefully load on a Ficoll/leucosep tube and centrifuge at 1,600 x g for 20 min, at room temperature.</li>
	<li>Collect monocytes at the interface into a new tube. Wash 3x with 10 ml 1x PBS containing 0.1% ethylenediaminetetraacetic acid (EDTA) by successive centrifugations at 1,000 x g, 370 x g and 160 x g for 10 min each, and then once in 1x PBS alone at 160 x g for 10 min.</li>
	<li>Resuspend the cell pellet in 5 ml RPMI-1640 medium without serum and seed the cells in 35 mm dishes at a density of 1 x 106 cells per dish.</li>
	<li>After sedimentation for 90 min in the incubator, discard the supernatant containing the non-adherent cells. Wash the adherent cells, consisting of monocytes, 3x with 1 ml PBS; then add 1 ml of fresh medium containing 10% (v/v) human serum to the previously serum free cells.</li>
	<li>After 6 days of culture, to yield alternative differentiated macrophages (M2), add recombinant human IL-4 (15 ng/ml) and maintain for 6 days. Then, treat differentiated macrophages with lipopolysaccharide (100 ng/ml) at day 12 for 4 hr to obtain M1 macrophages.</li>
</ol>
3. Extraction of M1 and M2 Macrophage Proteins for 2D Electrophoresis
<ol>
	<li>Wash macrophages three times with 25 mM Tris, pH 7.4, and scrap in buffer containing 30 mM Tris pH 8, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 2 M thiourea and 7 M urea.</li>
	<li>Lyse cells using a mixer suitable for 1.5 ml microcentrifuge tubes for 5 min in ice and store at -20 &#176;C.</li>
	<li>Determine protein concentration using a commercial Bradford reagent. Store 100 &#181;l aliquots of the proteins at -20 &#176;C until use.</li>
</ol>
4. Isoelectrofocusing
NOTE: Perform all labelling procedures in the dark.
<ol>
	<li>Reduce 5 &#181;g of each sample (M1 and M2 macrophages) adjusted to 9 &#956;l with lysis buffer with 2 mM Tris(2-carboxyethyl)phosphine (TCEP) for 1 hr at 37 &#176;C.</li>
	<li>Add cyanine 3 (Cy3) to M1 extract and M2 sulfhydryl-reactive dye extract at a concentration of 0.8 nM/&#956;g protein. Add cyanine 5 (Cy5) to M1 and M2 extracts at a concentration of 0.8 nM/&#956;g protein and incubate for 30 min at 37 &#176;C.</li>
	<li>Stop the reaction with the addition of an equal volume of sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 130 mM dithiothreitol (DTT) and 2% pharmalytes.</li>
	<li>Mix Cy3-labeled M1 samples with Cy5-labeled M2 samples in one hand and Cy3-labeled M2 samples with Cy5-labeled M1 samples in another hand.</li>
	<li>Rehydrate an Immobilized pH gradient (IPG) strip (240 mm, pH 3-10 linear gradient) with 450 &#956;l of labelled mixed samples in buffer containing 7 M urea, 2 M thiourea and 4% CHAPS on a isoelectric focusing (IEF) cell system for 24 hr without applying any current.</li>
	<li>Perform focusing at 300 volts (V) for 3 hr, and then at a gradient to 1,000 V for 6 hr, at a gradient to 8,000 V for 3 hr and finally at 8,000 V for 3 hr.</li>
</ol>
5. Second Dimension
NOTE: Perform all electrophoresis procedures in the dark.
<ol>
	<li>Incubate the IPG strips in equilibration buffer containing 0.1 mM Tris-HCl (pH 8), 6 M urea, 2% (w/v) sodium dodecyl sulfate (SDS) and 30% (v/v) glycerol for 10 min.</li>
	<li>Transfer the equilibrated IPG strips for the second dimension (SDS-polyacrylamide gel electrophoresis (PAGE)) onto 12.5% PAGE gels and seal with low-melting agarose.</li>
	<li>Carry out electrophoresis at 20 &#176;C using an Ettan-Daltsix system at a constant voltage of 70 V overnight followed by 300 V until the bromophenol blue front reaches the bottom of the gel.</li>
</ol>
6. Image Acquisition and Bioinformatic Analysis
<ol>
	<li>Scan gels cast between the two low-fluorescence glass plates with a DIGE Imager scanner at excitation/emission wavelengths of 532/580 nm for Cy3 and 633/670 nm for Cy5 to yield images with a pixel size of 100 &#956;m.</li>
	<li>Perform image analysis with commercial software as previously detailed12.</li>
	<li>Calculate and normalize spot volumes in each image. Assign a normalized spot volume as a proportion of the total value of each spot detected in the gel.</li>
	<li>Analyse the differences in protein spot volumes for each type of macrophages by comparing the normalized spot volume value between the two groups (M1 and M2). Consider the difference between the spot volumes to be significant if the change is 1.5 fold (p &#60;0.05, one-way ANOVA analysis).</li>
</ol>

<ol>
	<li>van Ginderachter, J. 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=Classical+and+alternative+activation+of+mononuclear+phagocytes:+picking+the+best+of+both+worlds+for+tumor+promotion.">Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion.</a> Immunobiology. 211, (2006).</li><li>Porcheray, F., 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+switching:+an+asset+for+the+resolution+of+inflammation.">Macrophage activation switching: an asset for the resolution of inflammation.</a> Clin. Exp. Immunol. 142, 481-489 (2005).</li><li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat. Rev. Immunol. 3, 23-35 (2003).</li><li>Mantovani, A., Locati, M., Vecchi, A., Sozzani, S., Allavena, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoy+receptors:+a+strategy+to+regulate+inflammatory+cytokines+and+chemokines.">Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines.</a> Trends Immunol. 22, 328-336 (2001).</li><li>Pinet, F., 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=Morphology,+homogeneity+and+functionality+of+human+monocytes-derived+macrophages.">Morphology, homogeneity and functionality of human monocytes-derived macrophages.</a> Cell. Mol. Biol. 49, 899-905 (2003).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+and+granulocytes+from+human+blood.">Isolation of mononuclear cells and granulocytes from human blood.</a> Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1. 97, 77-89 (1968).</li><li>Chinetti-Gbaguidi, G., 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=Human+atherosclerotic+plaque+alternative+macrophages+display+low+cholesterol+handling+but+high+phagocytosis+because+of+distinct+activities+of+the+PPAR&#947;+and+LXR&#945;+pathways.">Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPAR&#947; and LXR&#945; pathways.</a> Circ. Res. 108, 985-995 (2011).</li><li>Tonge, R., 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=Validation+and+development+of+fluorescence+two-dimensional+differential+gel+electrophoresis+proteomics+technology.">Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.</a> Proteomics. 1, 377-396 (2001).</li><li>Shaw, 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=Evaluation+of+saturation+labelling+two-dimensional+difference+gel+electrophoresis+fluorescent+dyes.">Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes.</a> Proteomics. 3, 1181-1195 (2003).</li><li>Dupont, 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=Application+of+saturation+dye+2D+DIGE+proteomics+to+characterize+proteins+modulated+by+oxidized+low+density+lipoprotein+treatment+of+human+macrophages.">Application of saturation dye 2D DIGE proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages.</a> J. Proteome Res. 7, 3572-3582 (2008).</li><li>Dupont, 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=Two-dimensional+maps+and+databases+of+the+human+macrophage+proteome+and+secretome.">Two-dimensional maps and databases of the human macrophage proteome and secretome.</a> Proteomics. 4, 1761-1778 (2004).</li><li>Acosta-Martin, A. E., 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=Impact+of+incomplete+DNase+I+treatment+on+human+macrophage+analysis.">Impact of incomplete DNase I treatment on human macrophage analysis.</a> Proteomics Clin. Appl. 3, 1236-1246 (2009).</li><li>Acosta-Martin, A. E., Lane, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+bioinformatics+and+MS-based+proteomics:+clinical+implications.">Combining bioinformatics and MS-based proteomics: clinical implications.</a> Expert Rev Proteomics. , (2014).</li><li>Boytard, 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=Role+of+proinflammatory+CD68+MR-+macrophages+in+peroxiredoxin-1+expression+and+in+abdominal+aortic+aneurysms+in+humans.">Role of proinflammatory CD68+MR- macrophages in peroxiredoxin-1 expression and in abdominal aortic aneurysms in humans.</a> Arterioscler. Thromb. Vasc. Biol. 33, 431-438 (2013).</li></ol>

There are no declared conflict of interest.

The protocol described herein details a method to analyse the impact of various stimuli of the two subtypes of macrophages, M1 (pro-inflammatory) and M2 (anti-inflammatory). Primary cultures of M1 and M2 macrophages were obtained from the differentiation of monocytes as previously published7.
The procedure of 2D DIGE gel electrophoresis requires specialized materials and equipment, such as IEF cell for isoelectrofocusing, low-fluorescence plates for the SDS-PAGE in order to scan twice the gel at excitation/emission wavelength for Cy3 and Cy5, a scanner for fluorescence and 2D software. This method requires some practice and manual dexterity.
There are several critical steps for successful 2D gel electrophoresis: 1) the extraction of proteins is critical in an environment without any contaminants such as albumin or keratin, and 2) performing the labelling and electrophoresis in the dark. Avoid the use of DTT for protein reduction in the lysis buffer as the technique requires the use of TCEP for reducing peptide sulfide bonds in proteins before labelling with cyanine reactive dyes.
This sensitive 2D DIGE technique allows the assessment of the differential expression of protein spots by macrophages under various environmental conditions, in the presence or absence of proteins depending on the subtype.
One of the limitations of this 2D DIGE technique is the need to label the proteins on the cysteine residue, which is absent in 14% of the protein. The other limitation is the relatively small number of samples that can be analysed at the same type in comparison to high throughput technology like quantitative mass spectrometry, though 2D DIGE is less costly.
Microbial stimuli, such as LPS as well as Th1 cytokines, activate macrophages into an M1 inflammatory state, while Th2 cytokines, such as IL-4, polarize cells to an M2 phenotype with immunoregulatory, repairing, and anti-inflammatory functions3. Phenotyping M1 and M2 cultured macrophages with or without host pathogenicity through proteomics approaches will provide a more detailed picture of the complex functional role of individual subtypes. The 2D DIGE electrophoresis may be helpful for the identification of polypeptides/proteins or post-translational modifications of these proteins differentially modified in one subtype or between the two subtypes of macrophages under specific stimuli or environment.

Macrophages are heterogeneous and plastic cells that are able to acquire distinct functional phenotypes. In vivo, these cells respond to a large variety of micro environmental signalssuch as microbial products, cytokines, etc.1. In vitro, the pro-inflammatory phenotype (M1) of macrophage can be induced by lipopolysaccharide (LPS) and the anti-inflammatory phenotype (M2) by some cytokines such as interleukin-4 (IL-4). Moreover, macrophages can switch from an activated M1 to M2 phenotype, and conversely, upon specific signals2.
Depending on the phenotype, macrophages will have different functions. M1 macrophages are cells that produce pro-inflammatory cytokines, such as tumor necrosis factor &#945; (TNF-&#945;), to kill microorganisms or tumor cells3. In contrast, M2 macrophages prevent these inflammatory response like in wound healing and fibrosis by producing anti-inflammatory factors such as TGF-&#946;3,4.
Human peripheral blood mononuclear cells from healthy donors were isolated by Ficoll density gradient centrifugation as previously described5 using a technique adapted from Boyum6. Macrophages in culture can be differentiated into M1 or M2 phenotype after 6 days of primary culture7.
Analysis of protein expression or protein changes between the two subtypes of macrophages under various controlled stimuli, such as host pathogenicity or microbial toxins, will be helpful to decipher the functionality of the pro- and anti-inflammatory macrophages.
Proteomics are unique tools for direct monitoring of proteins that are specifically up- or down-regulated in human cultured macrophages under various stimuli. Fluorescent dyes have resolved some of the limitations of 2D gel electrophoresis, such as low sensitivity and image analysis8. The dyes reacting with cysteine residues have increased the detection sensitivity compared to those reacting with lysine residues9. In a previous study, we demonstrated the usefulness of DIGE saturation labelling for the analysis of scarce samples10 compared to the classical silver-stained 2D electrophoresis11. This technology is helpful in rapidly analysing protein modifications between the two subtypes of macrophages or between untreated and treated macrophages from the same subtype.
The advantages of this proteomic technique are having access to the information of protein size and post-translational modifications by analysing the 2D gel12. It should be taken into account that this is not a high throughput technique, limiting the number of samples that can be analysed. The development of high throughput assays based on mass spectrometry as reviewed recently13 can improve this.
Here we present how to perform 2D DIGE analysis from the protein extraction of cultured macrophages through the processes of electrophoresis, isoelectrofocusing, and SDS-PAGE as well as information on the usefulness of adequate 2D software.

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>31870-074</td><td></td></tr><tr><td>PBX 10 X</td><td>Invitrogen</td><td>14200-083</td><td></td></tr><tr><td>L-glutamine-200 mM-100X</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>gentamycin 10 mg/ml</td><td>Invitrogen</td><td>15710-049</td><td></td></tr><tr><td>human serum</td><td>Invitrogen</td><td>34005100</td><td></td></tr><tr><td>Ficoll d = 1,077</td><td>ATGC</td><td>L6115</td><td></td></tr><tr><td>Leucosep</td><td>Dutscher</td><td>16760</td><td></td></tr><tr><td>&amp;nbsp;6-wells plate PRIMARIA&amp;nbsp;</td><td>Becton Dickinson</td><td>353846</td><td></td></tr><tr><td>IL-4</td><td>Promocell</td><td>B-61410</td><td></td></tr><tr><td>lipopolysaccharide</td><td>Sigma-Aldrich</td><td>L-2654</td><td></td></tr><tr><td>Giemsa</td><td>Fluka</td><td>48900</td><td></td></tr><tr><td>EDTA MM372,2</td><td>Research Organics&amp;nbsp;</td><td>3.00E+01</td><td></td></tr><tr><td>Filter 0.22 &amp;micro;m</td><td>Millipore</td><td>SCGPTORE</td><td></td></tr><tr><td>100 ml cylinder</td><td>Corning</td><td>430182</td><td></td></tr><tr><td>TCEP</td><td>Interchim</td><td>UP242214</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>Cy3+Cy5-reactive dye</td><td>GE Healthcare</td><td>25-8009-83</td><td></td></tr><tr><td>IPG strip 3-10 24cm</td><td>GE Healthcare</td><td>17-6002-44</td><td></td></tr><tr><td>Protean IEF cell</td><td>Bio-Rad</td><td>165-4000</td><td></td></tr><tr><td>Low-melting agarose</td><td>Invitrogen</td><td>15517-014</td><td></td></tr><tr><td>Ettan-Daltsix system</td><td>GE Healthcare</td><td>80-6485-08</td><td></td></tr><tr><td>Ettan DIGE Imager scanner</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Progenesis Samespot</td><td>Non linear dynamics</td><td></td><td></td></tr><tr><td>50 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>15 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>CHAPS</td><td>Sigma-Aldrich</td><td>C5070</td><td></td></tr><tr><td>Ur&amp;eacute;e</td><td>Merk</td><td>108484-500</td><td></td></tr><tr><td>Thiour&amp;eacute;e</td><td>Sigma-Aldrich</td><td>T7875</td><td></td></tr><tr><td>DTT</td><td>Bio-Rad</td><td>1610611</td><td></td></tr><tr><td>APS</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Pharmalytes 3-10</td><td>GE Healthcare</td><td>17-0456-01</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L3773</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114391</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279</td><td></td></tr><tr><td>Acrylamide 40%</td><td>Bio-Rad</td><td>161-0148</td><td></td></tr><tr><td>2D clean Up</td><td>GE Healthcare</td><td>80-6454-51</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Dim&amp;eacute;thylformamide</td><td>Sigma-Aldrich</td><td>22705-6</td><td></td></tr><tr><td>electrode wicks</td><td>Bio-Rad</td><td>165-4071</td><td></td></tr></tbody></table>

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.

To perform appropriate differential proteomic analysis, the processing of samples to be analysed should be verified.
In the example presented, the cell culture quality of macrophages is required for morphological and molecular aspects as previously published5. The differentiation of monocytes into macrophages and the homogeneity of the culture was followed by phase-microscopy. Figure 1 showed an example of primary cultures of M1 and M2 macrophages. As shown, we verified the homogeneity of M1 (Figure 1A, B) and M2 (Figure 1C, D) subtypes of macrophages at day 12 of culture by phase-microscopy at low (Figure 1A, C) and high (Figure 1B, D) magnification. Clearly, we observed a distinct morphology of macrophages M1 and M2 after 12 days of primary culture. As previously published, we verified by RT-PCR the presence of mRNA coding for TNF and IL1B in M1 macrophages and for MRC1 and CCL18 in M2 macrophages14 (not shown).
Only cultures combining those criteria were used for proteomic analysis.
We determined the 2D protein patterns of the two subtypes of macrophages, M1 (pro-inflammatory) and M2 (anti-inflammatory). For that purpose, we extracted proteins from M1 and M2 cultured macrophages as detailed in the protocol and M1 and M2 macrophages were either labelled with Cy3 or Cy5 saturation dyes to analyse the proteins by 2D DIGE electrophoresis. Figure 2 presents representative 2D gels of M1 (Figure 2A, B) and M2 (Figure 2C, D) macrophages of sufficient quality for bioinformatic analysis. The same pattern of proteins was observed for either M1 or M2 independently of the cyanine dyes used Cy3 (Figure 2A, C) or Cy5 (Figure 2B, D). In this example, a large linear pH gradient was used (3-10) and for detailing proteins with an acidic or basic pl, narrow pH gradient can be used.
Interestingly, the bioinformatic analysis of the 2D gel revealed 20 areas containing spots that can be analysed and compared using the 2D software. Figure 3 is an example of numerised 2D gel in which the 20 areas of spots are located. The 2D software is able to quantify and compare the volume of each spot between several 2D gels from different samples. The software can quantify the increase or decrease of spot volume and this can be visualized by colour as indicated in Figure 3. Spots are considered to have significant differential expression if the fold-change of normalized spot volumes as indicated in paragraph 6 of protocol section was greater than 1.5 with a p-value &lt;0.05. The presence or absence of a protein or polypeptide spot between the two groups of macrophages can be detected by the software.
<img alt="M1 and M2 macrophages comparison in microscope images showing morphological differences." fo:content-width="6in" src="/files/ftp_upload/52219/52219fig1highres.jpg" width="600" data-altupdatedat="1747929244000" data-alt="Figure 1"> 
Figure 1: Photographs of phase-contrast microscopy of primary culture of macrophages. Example of morphology of M1 (A,  B) and M2 (C,  D) macrophages. M1 macrophages were obtained by lipopolysaccharide treatment for 4 hr at day 12. M2 macrophages were obtained by IL-4 treatment starting from day 6 until day 12. Phase-contrast microscopy allows clear identification of both subtypes of macrophages. Photographs at low (10X) (A, C) and high (40X) (B, D) magnification were performed at day 12 of culture. Scale bar: 50 μm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52219/52219fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="2D electrophoresis protein expression comparison diagram, M1/M2 macrophages, Cy3/Cy5 labeling." fo:content-width="6in" src="/files/ftp_upload/52219/52219fig2highres.jpg" width="600" data-altupdatedat="1747929244000" data-alt="Figure 2"> 
Figure 2: Representative 2D DIGE gel of M1 and M2 cultured macrophages. Proteins (5 μg) were labelled with either Cy3 (A, B) or Cy5 (C, D) from M1 (A, B) and M2 (C, D) macrophages cultured for 12 days. The same pattern of 2D gel for M1 or M2 macrophages was obtained independently of the labelled cyanine used. The positions of molecular weight (Mr) standards are indicated on the left, and the pI is indicated on the bottom of the gel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52219/52219fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Proteomics analysis: annotated 2D electrophoresis result chart, showing protein expression changes." fo:content-width="6in" src="/files/ftp_upload/52219/52219fig3highres.jpg" width="600" data-altupdatedat="1747929244000" data-alt="Figure 3"> 
Figure 3: Bioinformatic analysis of a representative 2D DIGE gel from macrophage proteins with Progenesis SameSpots software. Twenty areas were detected to perform the differential analysis between M1 and M2 macrophages. The bottom of the figure indicates the color code for the fold-change of volume spots. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52219/52219fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

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 ...

proteomic-profiling-of-macrophages-by-2d-electrophoresis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.6K Views.  University Lille Nord de France. 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.

Video: Proteomic Profiling of Macrophages by 2D Electrophoresis

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Cancer Research

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Biology

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

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

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

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

Biochemistry

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Neuroscience

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Medicine

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

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. ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video