title: Chromatin immunoprecipitation for human monocyte derived macrophages.
abstract: The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a "one size fits all" rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM) [an acceptable in vitro model for primary, human macrophage cells], we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10min. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge.
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 985800 Nebraska Medical Center, Omaha, NE 68198-5800, United States.
authors.forename: Jessica
authors.initials: J
authors.lastname: Wooden
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 985800 Nebraska Medical Center, Omaha, NE 68198-5800, United States. Electronic address: pciborowski@unmc.edu.
authors.forename: Pawel
authors.initials: P
authors.lastname: Ciborowski
mesh_terms: D018929:Cell Culture Techniques; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D057890:Epigenomics / Q000379:methods; D006801:Humans; D008264:Macrophages; D016133:Polymerase Chain Reaction; D012984:Software; D013010:Sonication
keywords: ChIP; Histone modifications; MDM; Macrophage; Monocyte; UCSC Genome Browser