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Updated: May 12, 2026
Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells
Published on: March 8, 2024
Single-cell measurements of IgE-mediated FcεRI signaling using an integrated microfluidic platform
Yanli Liu1, Dipak Barua, Peng Liu
1Biotechnology and Bioengineering Department, Sandia National Laboratories, Livermore, California, United States of America.
Insights
Cellular responses vary, impacting overall health. This study introduces a microfluidic platform for single-cell analysis, revealing that protein variations drive signaling heterogeneity in allergic reactions.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Cellular response heterogeneity is crucial for immune function and disease outcomes.
- Traditional methods lack the single-cell resolution needed to study this heterogeneity.
- Microfluidics offers a promising avenue for high-resolution cellular analysis.
Purpose of the Study:
- To develop and validate a microfluidic platform for automated, high-throughput single-cell signaling analysis.
- To investigate the sources of heterogeneity in FcεRI signaling pathways.
- To quantify cell-by-cell variations in signaling events related to allergic responses.
Main Methods:
- Integration of microfluidics with on-chip flow cytometry and optical imaging for simultaneous measurements.
- Automated cell culture, stimulation, and sample preparation on a single device.
- Analysis of IgE receptor (FcεRI) signaling in RBL-2H3 cells, including protein phosphorylation and calcium mobilization.
Main Results:
- The platform enables quantitative, single-cell measurements of signaling events from small cell populations.
- Demonstrated ability to capture heterogeneous responses in FcεRI signaling.
- Model-based analysis identified protein copy number variations, specifically Lyn, as a key driver of Syk phosphorylation heterogeneity.
Conclusions:
- The developed microfluidic platform provides a powerful tool for dissecting cellular heterogeneity in signaling pathways.
- Understanding cell-to-cell variability in FcεRI signaling is critical for comprehending allergic reactions.
- Protein abundance directly influences signaling dynamics and contributes to observed cellular heterogeneity.
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.
