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Expression and translocation of fluorescent‐tagged p21‐activated kinase‐binding domain and PH domain of protein kinase B during murine neutrophil chemotaxis
Author(s) -
Magalhães Marco A. O.,
Zhu Fei,
Sarantis Helen,
GrayOwen Scott D.,
Ellen Richard P.,
Glogauer Michael
Publication year - 2007
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1189/jlb.0207126
Subject(s) - biology , microbiology and biotechnology , chemotaxis , transfection , green fluorescent protein , kinase , cell culture , biochemistry , receptor , gene , genetics
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 ∼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.