title: Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.
abstract: 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.
authors: Biotechnology and Bioengineering Department, Sandia National Laboratory, P.O. Box 969, Livermore, CA 94551, USA.
authors: Meiye
authors: M
authors: Wu
authors: Thomas D
authors: TD
authors: Perroud
authors: Nimisha
authors: N
authors: Srivastava
authors: Catherine S
authors: CS
authors: Branda
authors: Kenneth L
authors: KL
authors: Sale
authors: Bryan D
authors: BD
authors: Carson
authors: Kamlesh D
authors: KD
authors: Patel
authors: Steven S
authors: SS
authors: Branda
authors: Anup K
authors: AK
authors: Singh
mesh_terms: D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry; D007700:Kinetics; D008070:Lipopolysaccharides / Q000633:toxicity; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051193:Toll-Like Receptors / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; C498144:Rela protein, mouse; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3