(521d) A Microfludic Device For Flow Cytometric Analysis Of Innate Immunity Via TLR4 Signaling In Macrophage Cells

Microfluidics provides unique technologies for exploring unprecedented realms in complex, yet fundamental, cellular analysis. One such realm that we are investigating is host pathogen interactions with single cell resolution. Macrophage cells, a key component of innate immunity, respond to microbial pathogens through membrane proteins called Toll-Like Receptors (TLRs). Specifically, TLR4 surface receptors identify lipopolysaccharide (LPS) present on the cell envelope of Gram-negative bacteria (e.g., Francisella tularensis, Yersinia pestis). Our presentation will report on the development of an integrated device that conducts complex cell preparation as well as flow cytometric analysis for real-time monitoring of key processes in TLR4 signaling.

Flow Cytometry is a high throughput tool for interrogating single cell ? however, the different upstream cell handling steps involved in preparing cells for FC is a bottleneck. Sample addition, LPS dosage, mixing, multiple reagent addition for ? temporal fixation of proteins, permeabilization of the cell membrane, labeling with phospho-specific antibodies, and selective washing steps are required. Our microfluidic platform integrates necessary cell handling steps with on chip fluorescence-based flow cytometry. The fused silica microfluidic device integrates mixers (t~15s), incubation chambers (~1000 cells per assay), surface modification for low cell adhesion and hydrodynamic focusing for Flow Cytometric detection (~10 cells/min). Multiple exchange of reagents is enabled through in situ photopolymerized porous acrylate filters (pore size ~4µm) that act as size-exclusion filters ? allowing bulk pressure-driven fluid to flow through the monoliths, while retaining macrophages. Initial results from the assays are in agreement with conventional FC, with the compelling advantage of using only 10 µL of fluid and total analysis times of 15 min. We have also developed assays to detect the time-lapse phosphorylation of p38 (p38-P) and ERK (ERK-P), key proteins in TLR4 response. We will report detailed results from the streamlined flow cytometry assays and present details of our understanding of the innate immune response.