Proteomic Profiling of Macrophages by 2D Electrophoresis

Summary

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.

Abstract

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.

Introduction

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 functio....

Protocol

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°DC2010-1209).

1. Material and Culture Media Preparation

1. Dilute 10x Phosphate Buffer Saline (PBS) in sterile distilled water to obtain 1x PBS.
2. Make RPMI 1640 medium supplemented with gentamicin (40 μg/ml) and L-glutamine (2 mM), with and without 10% pooled human serum.

2. Primary Cultures of Monocyte-deri....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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