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.
Recommended Citation
Y. Liu et al., "Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform," PLoS ONE, vol. 8, no. 3, PLoS ONE, Mar 2013.
The definitive version is available at https://doi.org/10.1371/journal.pone.0060159
Department(s)
Chemical and Biochemical Engineering
Keywords and Phrases
Autacoid; Beta N Acetylhexosaminidase; Fc Epsilon Receptor; Fc Receptor; Immunoglobulin E Receptor; Protein Kinase Lyn; Protein Kinase Syk; Tumor Necrosis Factor Alpha; Unclassified Drug, Allergic Reaction; Animal Cell; Article; Autoanalysis; Calcium Mobilization; Calcium Transport; Cell Culture; Cell Population; Cell Stimulation; Complex Formation; Controlled Study; Copy Number Variation; Cytokine Production; Degranulation; Enzyme Phosphorylation; Enzyme Release; Flow Cytometry; Fluorescence Imaging; Microfluidic Analysis; Microfluidic Chip; Nonhuman; Protein Cross Linking; Protein Polymorphism; Protein Protein Interaction; Rat; Signal Transduction; Single Cell Analysis, Animals; Beta-N-Acetylhexosaminidases; Calcium; Cell Degranulation; Humans; Immunoglobulin E; Intracellular Signaling Peptides And Proteins; Microfluidics; Models, Immunological; Phosphorylation; Protein-Tyrosine Kinases; Rats; Receptors, IgE; Signal Transduction; Single-Cell Analysis; Time Factors; Tumor Necrosis Factor-Alpha
International Standard Serial Number (ISSN)
1932-6203
Document Type
Article - Journal
Document Version
Final Version
File Type
text
Language(s)
English
Rights
© 2013 PLoS ONE, All rights reserved.
Publication Date
01 Mar 2013
PubMed ID
23544131
Comments
This work was supported by National Institutes of Health/National Institute of General Medical Sciences grant P50GM085273. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.