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Originally published online as doi:10.1189/jlb.0207126 on May 29, 2007

Published online before print May 29, 2007
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(Journal of Leukocyte Biology. 2007;82:559-566.)
© 2007 by Society for Leukocyte Biology

Expression and translocation of fluorescent-tagged p21-activated kinase-binding domain and PH domain of protein kinase B during murine neutrophil chemotaxis

Marco A. O. Magalhães*, Fei Zhu*, Helen Sarantis{dagger}, Scott D. Gray-Owen{dagger}, Richard P. Ellen* and Michael Glogauer*,1

* CIHR Group in Matrix Dynamics and Dental Research Institute, Faculty of Dentistry, and
{dagger} Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada

1 Correspondence: 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. E-mail: michael.glogauer{at}utoronto.ca

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.

Key Words: transfection • PI-3K • Rac activation




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C. X. Sun, M. A.O. Magalhaes, and M. Glogauer
Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor
J. Cell Biol., October 22, 2007; 179(2): 239 - 245.
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