title: Analyzing cell physiology in C. elegans with fluorescent ratiometric reporters.
abstract: Ratiometric fluorescent reporters have recently emerged a new technique to non-invasively measure aspects of cell physiology such as redox status, calcium levels, energy production, and NADH levels. These reporters consist of either a single or pair of fluorophores along with specific modifications, such as the addition of a protein domain which binds to a metabolite of interest, thereby producing gradual alterations in fluorescence in response to changes in the measured parameter. Measurement of the changes in fluorescence produces a quantitative read-out of the cellular environment. While these reporters were initially developed to easily visualize and track changes in cultured cells, several groups have adapted these reporters to use in Caenorhabditis elegans which opens a new avenue through which to explore cell physiology during development or aging, in response to changes in external environment, or in response to genetic manipulation. These reporters have the advantage of being easily targeted to any part of the worm, and because C. elegans is transparent both the reporters and changes in their fluorescence can be clearly observed in vivo. Here we discuss the application of ratiometric reporters to C. elegans, and outline a method to quantitatively measure changes in intracellular peroxide levels using the HyPer ratiometric reporter. However, these principles can be applied to alternate ratiometric reporters which are designed to measure either other chemical species or other cellular parameters.
authors.affiliation: Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, UTHSCSA, San Antonio, TX 78229, United States; Center for Healthy Aging, UTHSCSA, San Antonio, TX 78229, United States.
authors.forename: Hongning
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, United States.
authors.forename: Uma
authors.initials: U
authors.lastname: Karadge
authors.affiliation: B&B Microscopes, 490 Lowries Run Road, Pittsburgh, PA 15237, United States.
authors.forename: William H
authors.initials: WH
authors.lastname: Humphries
authors.affiliation: Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, UTHSCSA, San Antonio, TX 78229, United States; Center for Healthy Aging, UTHSCSA, San Antonio, TX 78229, United States; GRECC, South Texas VA Healthcare System, San Antonio, TX 78229, United States. Electronic address: fishera2@uthscsa.edu.
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Fisher
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002468:Cell Physiological Phenomena / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D010545:Peroxides / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010545:Peroxides; D049452:Green Fluorescent Proteins; D002118:Calcium
keywords: Caenorhabditis elegans; Digital imaging; Fluorescent proteins; Metabolism; Ratiometric reporters; Transgene