Studies of Phosphorylation During Innate Immune Signaling using On-Chip Cell Preparation and Downstream Flow Cytometry

Fine temporal resolution is required for monitoring protein phosphorylation events key to cellular signaling pathways. With the goal of teasing apart the kinetics of phosphorylation cascades central to the human innate immune response to pathogen invasion, our group has developed a microfluidic device that integrates flow cytometry with required upstream cell preparation steps. Streamlined cell preparation and analysis allows monitoring of events with kinetic resolution on the order of minutes - not tens of minutes to hours. The planar microfluidic device contains 50mm long spiral mixers, porous polymer monoliths for selective exchange of reagents, and incubation chambers (~1000 cells per chamber) where macrophage cells (RAW264.7) are challenged with a chemical signal of Gram-negative bacteria (lipopolysaccharide) and subsequently labeled with fluorescent immunoreagents. Finally, on the same device, the labeled macrophage cells are analyzed using two-color flow cytometry. Such an integrated self-contained microfluidic platform promises to be of widespread use to host-pathogen studies in infectious disease laboratories.