title: Expression and translocation of fluorescent-tagged p21-activated kinase-binding domain and PH domain of protein kinase B during murine neutrophil chemotaxis.
abstract: Neutrophils are key cells of the innate immune system; they are terminally differentiated and therefore difficult to genetically manipulate and study in vitro. In the present study, we describe a protocol to transiently express two fluorescent markers, the PH domain of protein kinase B fused to red fluorescent protein and the p21-activated kinase-binding domain fused to a yellow fluorescent protein, in primary neutrophils. Using this approach, we are able to achieve a transfection efficiency of approximately 30%. The expression of the transfected probes occurred within 2 h and allowed for real-time monitoring of intermediates in key neutrophil activation pathways at the leading edge of migrating cells. We describe here a transfection protocol for primary neutrophils, which preserves fMLP-mediated cell polarization and cytoskeleton reorganization with simultaneous accumulation of PI-3K products and active Rac at the leading edge. The visualization and analysis of transfected fluorescent markers in primary neutrophils are a powerful technique to monitor chemotaxis signaling pathways in real time.
authors.affiliation: CIHR Group in Matrix Dynamics and Dental Research Institute, Faculty of Dentistry, University of Toronto, 241 Fitzgerald Building, 150 College Street, Toronto, Canada M5S 3E2.
authors.forename: Marco A O
authors.initials: MA
authors.lastname: Magalhães
authors.forename: Fei
authors.initials: F
authors.lastname: Zhu
authors.forename: Helen
authors.initials: H
authors.lastname: Sarantis
authors.forename: Scott D
authors.initials: SD
authors.lastname: Gray-Owen
authors.forename: Richard P
authors.initials: RP
authors.lastname: Ellen
authors.forename: Michael
authors.initials: M
authors.lastname: Glogauer
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D044042:Receptors, Formyl Peptide / Q000235:genetics / Q000378:metabolism; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D044042:Receptors, Formyl Peptide; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins; D000097573:Red Fluorescent Protein