Published online before print May 29, 2007
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Article |
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*CIHR Group in Matrix Dynamics and Dental Research Institute, Faculty of Dentistry, and
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.
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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