Name: proteomic profiling of macrophages by 2d electrophoresis
Uploaded: 2014-11-04T15:00:00
Description: Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

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 ...

<ol>
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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 twi...

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.

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		<tr height="17">
			<td height="17" style="height:17px;width:208px;"><a name="RANGE!A1:C43">Name</a></td>
			<td style="width:155px;">Company</td>
			<td style="width:111px;">Catalog number</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640</td>
			<td>Invitrogen</td>
			<td>31870-074</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBX 10 X</td>
			<td>Invitrogen</td>
			<td>14200-083</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine-200mM-100X</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">gentamycin 10 mg/mL</td>
			<td>Invitrogen</td>
			<td>15710-049</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">human serum</td>
			<td>Invitrogen</td>
			<td>34005100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ficoll d=1,077</td>
			<td>ATGC</td>
			<td>L6115</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Leucosep</td>
			<td>Dutscher</td>
			<td>16760</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;6-wells plate PRIMARIA&#160;</td>
			<td>Becton Dickinson</td>
			<td>353846</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IL-4</td>
			<td>Promocell</td>
			<td>B-61410</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">lipopolysaccharide</td>
			<td>Sigma-Aldrich</td>
			<td>L-2654</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Giemsa</td>
			<td>Fluka</td>
			<td>48900</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EDTA MM372,2</td>
			<td>Research Organics&#160;</td>
			<td>3.00E+01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter 0,22&#181;m</td>
			<td>Millipore</td>
			<td>SCGPTORE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100 mL cylinder</td>
			<td>Corning</td>
			<td>430182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TCEP</td>
			<td>Interchim</td>
			<td>UP242214</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bradford reagent</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3+Cy5-reactive dye</td>
			<td>GE Healthcare</td>
			<td>25-8009-83</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IPG strip 3-10 24cm</td>
			<td>GE Healthcare</td>
			<td>17-6002-44</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean IEF cell</td>
			<td>Bio-Rad</td>
			<td>165-4000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Low-melting agarose</td>
			<td>Invitrogen</td>
			<td>15517-014</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan-Daltsix system</td>
			<td>GE Healthcare</td>
			<td>80-6485-08</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan DIGE Imager scanner</td>
			<td>GE Healthcare</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Progenesis Samespot</td>
			<td>Non linear dynamics</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CHAPS</td>
			<td>Sigma-Aldrich</td>
			<td>C5070</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ur&#233;e</td>
			<td>Merk</td>
			<td>108484-500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Thiour&#233;e</td>
			<td>Sigma-Aldrich</td>
			<td>T7875</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DTT</td>
			<td>Bio-Rad</td>
			<td>1610611</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">APS</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pharmalytes 3-10</td>
			<td>GE Healthcare</td>
			<td>17-0456-01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L3773</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>114391</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G6279</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acrylamide 40%</td>
			<td>Bio-Rad</td>
			<td>161-0148</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2D clean Up</td>
			<td>GE Healthcare</td>
			<td>80-6454-51</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dim&#233;thylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>22705-6</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">electrode wicks</td>
			<td>Bio-Rad</td>
			<td>165-4071</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 veri...

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 ...

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