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

• Published in: Lab on a chip Vol. 12; no. 16; pp. 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: Journal Article
• Language: English
• Published: England 21.08.2012
• 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
• ISSN: 1473-0197; 1473-0189; 1473-0189
• DOI: 10.1039/c2lc40344g

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.