Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resoluti...

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Bibliographic Details
Published in:Lab on a chip 2012-08, Vol.12 (16), p.2823-2831
Main Authors: Wu, Meiye, Perroud, Thomas D, Srivastava, Nimisha, Branda, Catherine S, Sale, Kenneth L, Carson, Bryan D, Patel, Kamlesh D, Branda, Steven S, Singh, Anup K
Format: Article
Language:English
Subjects:
Animals
Cell culture
Cell Line
Flow Cytometry
Kinetics
Lipopolysaccharides - toxicity
Macrophages - drug effects
Macrophages - immunology
Macrophages - metabolism
Mice
Microfluidic Analytical Techniques - instrumentation
Microfluidic Analytical Techniques - methods
Mitogen-Activated Protein Kinase 1 - metabolism
Mitogen-Activated Protein Kinase 3 - metabolism
Phosphorylation
Signal Transduction - drug effects
Toll-Like Receptor 4 - metabolism
Toll-Like Receptors - metabolism
Transcription Factor RelA - metabolism
Tumor Necrosis Factor-alpha - metabolism
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Summary:We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.
ISSN:1473-0197
1473-0189
DOI:10.1039/c2lc40344g