title: A comparative analysis of standard microtiter plate reading versus imaging in cellular assays.
abstract: We evaluated the performance of two plate readers (the Beckman Coulter [Fullerton, CA] DTX and the PerkinElmer [Wellesley, MA] EnVision) and a plate imager (the General Electric [Fairfield, CT] IN Cell 1000 Analyzer) in a primary fluorescent cellular screen of 10,000 Molecular Libraries Screening Center Network library compounds for up- and down-regulation of vascular cell adhesion molecule (VCAM)-1, which has been shown to be up-regulated in atherothrombotic vascular disease and is a general indicator of chronic inflammatory disease. Prior to screening, imaging of a twofold, six-step titration of fluorescent cells in a 384-well test plate showed greater consistency, sensitivity, and dynamic range of signal detection curves throughout the detection range, as compared to the plate readers. With the same 384-well test plate, the detection limits for fluorescent protein-labeled cells on the DTX and EnVision instruments were 2,250 and 560 fluorescent cells per well, respectively, as compared to 280 on the IN Cell 1000. During VCAM screening, sensitivity was critical for detection of antagonists, which reduced brightness of the primary immunofluorescence readout; inhibitor controls yielded Z' values of 0.41 and 0.16 for the IN Cell 1000 and EnVision instruments, respectively. The best 1% of small molecule inhibitors from all platforms were visually confirmed using images from the IN Cell 1000. The EnVision and DTX plate readers mutually identified approximately 57% and 21%, respectively, of the VCAM-1 inhibitors visually confirmed in the IN Cell best 1% of inhibitors. Furthermore, the plate reader hits were largely exclusive, with only 6% agreement across all platforms (three hits out of 47). Taken together, the imager outperformed the plate readers at hit detection in this bimodal assay because of superior sensitivity and had the advantage of speeding hit confirmation during post-acquisition analysis.
authors.affiliation: Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Bushway
authors.forename: Mark
authors.initials: M
authors.lastname: Mercola
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Price
mesh_terms: D001331:Automation; D002465:Cell Movement / Q000502:physiology; D004396:Coloring Agents; D015536:Down-Regulation; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D015854:Up-Regulation; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis
chemical_list: D004396:Coloring Agents; D007202:Indicators and Reagents; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1
references.citation: J Biol Chem. 1996 Jan 26;271(4):1909-15
references.pmid: 8567638
references.citation: Assay Drug Dev Technol. 2005 Oct;3(5):483-99
references.pmid: 16305306
references.citation: Methods Enzymol. 2006;414:300-16
references.pmid: 17110199
references.citation: J Microsc. 2007 May;226(Pt 2):121-32
references.pmid: 17444941
references.citation: Assay Drug Dev Technol. 2007 Feb;5(1):29-48
references.pmid: 17355198
references.citation: J Biol Chem. 1998 Oct 30;273(44):28897-905
references.pmid: 9786892
references.citation: J Virol. 2000 Nov;74(22):10778-84
references.pmid: 11044122
references.citation: J Biomol Screen. 2006 Oct;11(7):864-9
references.pmid: 16973922
references.citation: Immunology. 1996 Apr;87(4):573-80
references.pmid: 8675211
references.citation: J Cell Biochem. 2006 Jul 1;98(4):770-88
references.pmid: 16440331
references.citation: Hum Gene Ther. 2000 Jan 1;11(1):179-90
references.pmid: 10646649