Accuri C6 Flow Cytometer System
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Published online before print May 29, 2007
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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0207126

Received for publication February 24, 2007.
Revised April 10, 2007.
Accepted for publication April 30, 2007.

Article

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 *@

*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

@ To whom correspondence should be addressed. E-mail: michael.glogauer{at}utoronto.ca.


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Abstract

Neutrophils are key cells of the innate immune system, which 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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