title: A mechanistically relevant cytotoxicity assay based on the detection of cellular GFP.
abstract: Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.
authors.affiliation: Cell Systems Science Group/Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. michael.halter@nist.gov
authors.forename: Michael
authors.initials: M
authors.lastname: Halter
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Almeida
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tona
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Cole
authors.forename: Anne L
authors.initials: AL
authors.lastname: Plant
authors.forename: John T
authors.initials: JT
authors.lastname: Elliott
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004396:Coloring Agents; D003603:Cytotoxins / Q000633:toxicity*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry*; D019044:Image Cytometry; D007091:Image Processing, Computer-Assisted; D012270:Ribosomes / Q000187:drug effects; D012276:Ricin / Q000633:toxicity; D013050:Spectrometry, Fluorescence; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection; D014709:Vero Cells
chemical_list: D004396:Coloring Agents; D003603:Cytotoxins; D013778:Tetrazolium Salts; D013844:Thiazoles; D049452:Green Fluorescent Proteins; D012276:Ricin; C022616:thiazolyl blue