Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

Abstract

Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. Here, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chip flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn.

IL-33 fine tunes mast cell degranulation and chemokine production at the single-cell level

“…We can hypothesize that important housekeeping cellular mechanisms or biochemical processes might interfere with cellular effector functions, such as degranulation or chemokine production. In this line heterogeneity in the mast cell degranulatory response was reported by Liu et al 57 by using an integrated microfluidic platform. The authors suggested that heterogeneity might stem from cell-to-cell variability in the copy number of the tyrosine kinase Lyn.…”

Timescale Separation of Positive and Negative Signaling Creates History-Dependent Responses to IgE Receptor Stimulation

“…Mouse monoclonal anti-DNP-IgE was prepared as described by Liu et al . 37 . Trivalent ligand DF3 and FAM-conjugated DF3 were custom synthesized by AnaSpec Inc. (Fremont, CA).…”

A Critical Comparison of Rejection-Based Algorithms for Simulation of Large Biochemical Reaction Networks

“…We use the high-affinity IgE receptor, referred to as FcǫRI, model developed by Liu et al [43] to show the performance of algorithms on the large biological model. FcǫRI is a high-affinity receptor for the antigen-specific immunoglobulin E (IgE).…”