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

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. In this paper, 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. Finally, model-based analysis of the Syk phosphorylation data suggestsmore » 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.« less
Authors:
 [1] ;  [2] ;  [1] ;  [3] ;  [3] ;  [2] ;  [1]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States). Biotechnology and Bioengineering Dept.
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Theoretical Biology and Biophysics Group. Theoretical Division. Center for Nonlinear Studies
  3. Univ. of New Mexico, Albuquerque, NM (United States). Dept. of Pathology. Cancer Center
Publication Date:
OSTI Identifier:
1321718
Report Number(s):
LA-UR--12-23884
Journal ID: ISSN 1932-6203
Grant/Contract Number:
AC52-06NA25396; AC04-94AL85000; P50GM085273
Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 8; Journal Issue: 3; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Research Org:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Sponsoring Org:
USDOE; National Inst. of Health (NIH) (United States)
Contributing Orgs:
Univ. of New Mexico, Albuquerque, NM (United States)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES biological science; phosphorylation; microfluidics; cytokines; protein kinase signaling cascade; flow cytometry; cell degranulation; fluorescence imaging; enzyme-linked immunoassays