title: Phenotyping CD4+ hTh2 Cells by Flow Cytometry: Simultaneous Detection of Transcription Factors, Secreted Cytokines, and Surface Markers.
abstract: Flow cytometry is a powerful technique that allows simultaneous detection of multiple markers on a specific cell population. This method is virtually unlimited as long as the specimen of interest can be put into a single-cell suspension for staining and subsequent analysis by the flow cytometer. Most investigators using this methodology are doing so because their cell population is rare in frequency and requires multiple markers to characterize their population of interest; thus standard methods such as Western blot and IHC are unsuitable due to limitations in cell number and the number of markers available. Most investigators using this method are using 6-14 parameters to study their cell populations of interest: however, using a large number of fluorochrome-labeled antibodies is hampered by the fact that suboptimal fluorochromes must be used, and that high and low cell density markers must be chosen with care. This is further complicated when the cell markers of interest are cytokines, transcription factors, surface markers, and/or phosphorylated proteins, each potentially requiring a specialized buffer system for optimal detection of the antibody of interest. This chapter focuses on optimizing flow cytometry staining methods for simultaneous detection of surface markers, transcription factors, secreted cytokines, and phosphorylated antibodies in a single stain on CD4+ human Th2 cells.
authors.affiliation: Bio-Techne, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Goetz
authors.affiliation: Bio-Techne, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413, USA.
authors.forename: Li-Jen
authors.initials: LJ
authors.lastname: Peng
authors.affiliation: Bio-Techne, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413, USA.
authors.forename: Birte
authors.initials: B
authors.lastname: Aggeler
authors.affiliation: Bio-Techne, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413, USA. Jody.Bonnevier@bio-techne.com.
authors.forename: Jody
authors.initials: J
authors.lastname: Bonnevier
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism; D015415:Biomarkers; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008213:Lymphocyte Activation; D010766:Phosphorylation; D018418:Th2 Cells / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D015415:Biomarkers; D016207:Cytokines; D014157:Transcription Factors
keywords: CCR3; CD4; Flow cytometry; GATA-3; IL-10; Peripheral blood mononuclear cells (PBMCs); Phospho-STAT6; Phospho-antibodies; T helper 2 cells (Th2); Th2 cytokines