title: Tools for Single-Cell Kinetic Analysis of Virus-Host Interactions.
abstract: Measures of cellular gene expression or behavior, when performed on individual cells, inevitably reveal a diversity of behaviors and outcomes that can correlate with normal or diseased states. For virus infections, the potential diversity of outcomes are pushed to an extreme, where measures of infection reflect features of the specific infecting virus particle, the individual host cell, as well as interactions between viral and cellular components. Single-cell measures, while revealing, still often rely on specialized fluid handling capabilities, employ end-point measures, and remain labor-intensive to perform. To address these limitations, we consider a new microwell-based device that uses simple pipette-based fluid handling to isolate individual cells. Our design allows different experimental conditions to be implemented in a single device, permitting easier and more standardized protocols. Further, we utilize a recently reported dual-color fluorescent reporter system that provides dynamic readouts of viral and cellular gene expression during single-cell infections by vesicular stomatitis virus. In addition, we develop and show how free, open-source software can enable streamlined data management and batch image analysis. Here we validate the integration of the device and software using the reporter system to demonstrate unique single-cell dynamic measures of cellular responses to viral infection.
authors.affiliation: Systems Biology Theme, Wisconsin Institute for Discovery, Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States of America.
authors.forename: Jay W
authors.initials: JW
authors.lastname: Warrick
authors.affiliation: Systems Biology Theme, Wisconsin Institute for Discovery, Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Timm
authors.affiliation: Systems Biology Theme, Wisconsin Institute for Discovery, Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States of America.
authors.forename: Adam
authors.initials: A
authors.lastname: Swick
authors.affiliation: Systems Biology Theme, Wisconsin Institute for Discovery, Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Yin
mesh_terms: D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000295:instrumentation*; D054884:Host-Pathogen Interactions / Q000235:genetics*; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism; D050839:Interferon Regulatory Factor-7 / Q000235:genetics / Q000378:metabolism; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D011467:Prostate / Q000378:metabolism / Q000473:pathology / Q000821:virology; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011971:Receptors, Immunologic; D015398:Signal Transduction; D059010:Single-Cell Analysis; D012984:Software; D018116:Vesiculovirus / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; D007370:Interferon Type I; D008164:Luminescent Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011971:Receptors, Immunologic; D000097573:Red Fluorescent Protein; C484289:IFIT2 protein, human; C494232:IRF3 protein, human; C494234:IRF7 protein, human; C452820:RIGI protein, human