title: Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.
abstract: Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f(-)/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology.
authors.affiliation: Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Gizem
authors.initials: G
authors.lastname: Turaç
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hindley
authors.forename: Ria
authors.initials: R
authors.lastname: Thomas
authors.forename: Jason A
authors.initials: JA
authors.lastname: Davis
authors.forename: Michela
authors.initials: M
authors.lastname: Deleidi
authors.forename: Thomas
authors.initials: T
authors.lastname: Gasser
authors.forename: Erdal
authors.initials: E
authors.lastname: Karaöz
authors.forename: Jan
authors.initials: J
authors.lastname: Pruszak
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D015703:Antigens, CD; D015415:Biomarkers