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

Neutrophils are key cells of the innate immune system, which are terminally differenti- ated and therefore difficult to genetically manipu- late and study in vitro. In the present study, we describe a protocol to transiently express two flu- orescent 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 trans- fected probes occurred within 2 h and allowed for real-time monitoring of intermediates in key neu- trophil 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 fluo- rescent markers in primary neutrophils are a pow- erful technique to monitor chemotaxis signaling pathways in real time. J. Leukoc. Biol. 82: 000-000; 2007.