title: Phospho-Flow Analysis of Primary Mouse Cells After HDAC Inhibitor Treatment.
abstract: Flow cytometric techniques allow fast, sensitive, and multiparametric analyses at the single cell level. This makes it possible to distinguish subsets of cells within heterogeneous samples. Moreover, flow cytometry has become a frequently used method for the evaluation of therapeutic effects. Here, we describe the analysis of the phosphorylation status of signal transducer and activator of transcription-1 (STAT-1) in primary mouse cells after treatment with histone deacetylase inhibitors (HDACi) that are currently considered anticancer agents. We provide detailed protocols for the preparation of murine bone marrow cells and the staining of HDACi-treated cells, as well as an insight into the concepts of flow cytometry analysis.
authors.affiliation: Zentrum für Infektiologie, Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Hildebrand
authors.affiliation: Zentrum für Infektiologie, Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany. kubatzky@uni-heidelberg.de.
authors.forename: Katharina F
authors.initials: KF
authors.lastname: Kubatzky
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001549:Benzamides / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D015870:Gene Expression; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D061251:Primary Cell Culture; D011725:Pyridines / Q000494:pharmacology*; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D001549:Benzamides; D056572:Histone Deacetylase Inhibitors; D011725:Pyridines; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; C118739:entinostat; D007371:Interferon-gamma; D006655:Histone Deacetylases; D016650:Fluorescein-5-isothiocyanate
keywords: Bone marrow-derived cells; Fluorescence cytometry; HDACi; Intracellular staining; Phospho-flow analysis