title: Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.
abstract: Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.
authors.affiliation: Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg.
authors.forename: Vishal
authors.initials: V
authors.lastname: Menon
authors.affiliation: Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg; Spemann Graduate School of Biology and Medicine and Faculty of Biology, University of Freiburg.
authors.forename: Ria
authors.initials: R
authors.lastname: Thomas
authors.affiliation: Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg; School of Life Sciences, Keele University.
authors.forename: Arun R
authors.initials: AR
authors.lastname: Ghale
authors.affiliation: Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg.
authors.forename: Christina
authors.initials: C
authors.lastname: Reinhard
authors.affiliation: Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg; Center for Biological Signaling Studies (BIOSS), University of Freiburg; jan.pruszak@anat.uni-freiburg.de.
authors.forename: Jan
authors.initials: J
authors.lastname: Pruszak
mesh_terms: D015703:Antigens, CD / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D058953:Neural Stem Cells / Q000737:chemistry / Q000166:cytology / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D015703:Antigens, CD; D000954:Antigens, Surface; D005452:Fluoresceins; D018796:Immunoconjugates; D007135:Immunoglobulin Variable Region; C024098:6-carboxyfluorescein