title: CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses.
abstract: Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.
authors.forename: Marco
authors.initials: M
authors.lastname: Fritzsche
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Fernandes
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.
authors.forename: Huw
authors.initials: H
authors.lastname: Colin-York
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Santos
authors.affiliation: Department of Chemistry, University of Cambridge, CB2 1EW Cambridge, United Kingdom.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Lee
authors.affiliation: Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.
authors.forename: B Christoffer
authors.initials: BC
authors.lastname: Lagerholm
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.|Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D019169:Jurkat Cells; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*; D012984:Software; D013601:T-Lymphocytes / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; D002118:Calcium