(166e) A Microfluidic Platform for Studying Host-Pathogen Interactions with Single-Cell Resolution

Innate immunity is our first line of defense against a pathogenic bacteria or virus. A comprehensive ?system-level? understanding of innate immunity pathways is the key to deciphering mechanisms of pathogenesis and may lead to improvements in early diagnosis or developing improved therapeutics. Current methods for studying signaling focus on measurements of a limited number of components in a pathway and hence, fail to provide a systems-level understanding. Current methods also lack single-cell resolution necessary for accurate quantification of concentration and kinetics. We have developed a systems biology approach to decipher toll-like receptor 4 (TLR4) pathways in murine macrophages in response to exposure to pathogenic bacteria and their lipopolysaccharide (LPS). Our approach integrates a microfluidic cell handling and analysis platform with high-resolution imaging to provide spatially- and temporally-resolved measurement of signaling pathways. The integrated microfluidic platform is capable of imaging single cells to obtain dynamic translocation data as well as high-throughput acquisition of quantitative protein expression and phosphorylation information of selected cell populations. The platform consists of multiple modules such as single-cell array, cell sorter, and phosphoflow chip to provide confocal imaging, cell sorting, and flow cytometry-based phosphorylation assays. The single-cell array module contains fluidic constrictions designed to trap and monitor up to 100 single cells for hours (cells viable for >72hrs), enabling detailed statistically-significant measurements. The module was used to analyze translocation behavior of transcription factor NF-kB in macrophages upon activation by E. coli and Y. pestis LPS. Activation of NF-kB is preceded by phosphorylation of many kinases and to correlate the kinase activity with translocation, we performed flow cytometry-based phosphorylation assays in the PhosphoChip module. To allow further downstream analysis on selected cells, we also implemented an optical-trapping based sorting of cells. This has allowed us to sort macrophages infected with bacteria from uninfected cells with the goal of obtaining data only on the infected (the desired) population. The various microfluidic chip modules have been integrated into a portable package that can be mounted on a typical inverted microscope. The microfluidic platform allows high-resolution imaging as well as quantitative proteomic measurements with high sensitivity (