title: Flow cytometric analysis of cytokine receptor signal transduction.
abstract: Cytokines are critical regulators of the development and maturation of hematopoietic cells. Signal transduction via cytokine receptors proceeds through activation of the JAK-STAT pathway to stimulate cell proliferation, differentiation and effector functions. Phosphorylation of intracellular STAT molecules by the receptor-associated JAK kinases is one of the very early events following cytokine stimulation. Western blot detection of tyrosine phosphorylated STAT molecules is widely used as a hallmark of cytokine receptor activation. However, this is not feasible when cells of interest are limiting, or represent a small fraction within a mixed population of different cell types. To circumvent this technical obstacle, we have developed techniques to detect phosphorylated STAT molecules in fixed cells by flow cytometry. The fixation and permeabilization protocols preserve the antigenicity of cell surface markers allowing us to distinguish distinct cell populations responding to cytokine stimulation. In this report, we demonstrate the use of this technique to rapidly and reliably identify, and quantify thymocyte subsets activated by interleukin-7. We envisage that this technique will find wide application in studying cytokine receptor signal transduction, particularly in identifying cytokine-dependent developmental checkpoints during hematopoiesis.
authors.affiliation: Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, ON, Canada M5G 2M9.
authors.forename: Subburaj
authors.initials: S
authors.lastname: Ilangumaran
authors.forename: Dina
authors.initials: D
authors.lastname: Finan
authors.forename: Robert
authors.initials: R
authors.lastname: Rottapel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002455:Cell Division; D004268:DNA-Binding Proteins / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods; D007376:Interleukin-2 / Q000378:metabolism; D015848:Interleukin-5 / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D018121:Receptors, Cytokine / Q000378:metabolism*; D020395:Receptors, Interleukin-7 / Q000378:metabolism; D050794:STAT1 Transcription Factor; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology*; D016176:T-Lymphocyte Subsets; D015534:Trans-Activators / Q000032:analysis
chemical_list: D004268:DNA-Binding Proteins; D007376:Interleukin-2; D015848:Interleukin-5; D019253:Proto-Oncogene Proteins c-bcl-2; D018121:Receptors, Cytokine; D020395:Receptors, Interleukin-7; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D015534:Trans-Activators; D020929:Mitogen-Activated Protein Kinase Kinases