title: Fluorescent protein-based cellular assays analyzed by laser-scanning microplate cytometry in 1536-well plate format.
abstract: Microtiter plate readers have evolved from photomultiplier and charged-coupled device-based readers, where a population-averaged signal is detected from each well, to microscope-based imaging systems, where cellular characteristics from individual cells are measured. For these systems, speed and ease of data analysis are inversely proportional to the amount of data collected from each well. Microplate laser cytometry is a technology compatible with a 1536-well plate format and capable of population distribution analysis. Microplate cytometers such as the Acumen Explorer can monitor up to four fluorescent signals from single objects in microtiter plates with densities as high as 1536 wells. These instruments can measure changes in fluorescent protein expression, cell shape, or simple cellular redistribution events such as cytoplasmic to nuclear translocation. To develop high-throughput screening applications using laser-scanning microplate cytometry, we used green fluorescent protein- and yellow fluorescent protein-expressing cell lines designed to measure diverse biological functions such as nuclear translocation, epigenetic signaling, and G protein-coupled receptor activation. This chapter illustrates the application of microplate laser cytometry to these assays in a manner that is suitable for screening large compound collections in high throughput.
authors.affiliation: NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Johnson
authors.forename: Ya-qin
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Henrike
authors.initials: H
authors.lastname: Veith
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.forename: Adam
authors.initials: A
authors.lastname: Yasgar
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.forename: Elisabeth D
authors.initials: ED
authors.lastname: Martinez
authors.forename: John K
authors.initials: JK
authors.lastname: Westwick
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: James
authors.initials: J
authors.lastname: Inglese
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D045744:Cell Line, Tumor; D003584:Cytological Techniques / Q000379:methods*; D003592:Cytophotometry / Q000379:methods; D044127:Epigenesis, Genetic; D005434:Flow Cytometry / Q000379:methods*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007834:Lasers; D008024:Ligands; D051379:Mice; D011965:Receptors, Glucocorticoid / Q000737:chemistry; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D008024:Ligands; D011965:Receptors, Glucocorticoid; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins