title: Proteomic profiling of macrophages by 2D electrophoresis.
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.
authors.affiliation: Inserm UMR744, University Lille Nord de France.
authors.forename: Marion
authors.initials: M
authors.lastname: Bouvet
authors.affiliation: Inserm UMR744, University Lille Nord de France.
authors.forename: Annie
authors.initials: A
authors.lastname: Turkieh
authors.affiliation: Inserm UMR744, University Lille Nord de France.
authors.forename: Adelina E
authors.initials: AE
authors.lastname: Acosta-Martin
authors.affiliation: Inserm UMR744, University Lille Nord de France.
authors.forename: Maggy
authors.initials: M
authors.lastname: Chwastyniak
authors.affiliation: Inserm UMR744, University Lille Nord de France.
authors.forename: Olivia
authors.initials: O
authors.lastname: Beseme
authors.affiliation: Inserm UMR744, University Lille Nord de France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Amouyel
authors.affiliation: Inserm UMR744, University Lille Nord de France; florence.pinet@pasteur-lille.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Pinet
mesh_terms: D002454:Cell Differentiation / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D008264:Macrophages / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins