title: A versatile assay for the accurate, time-resolved determination of cellular viability.
abstract: A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.
authors.affiliation: Department of Biology and Geoscience, Faculty of Science, Shizuoka University, 422-8529, Shizuoka, Japan. sbtaman@ipc.shizuoka.ac.jp
authors.forename: Toyoki
authors.initials: T
authors.lastname: Amano
authors.forename: Ken ichi
authors.initials: Ki
authors.lastname: Hirasawa
authors.forename: Michael J
authors.initials: MJ
authors.lastname: O'Donohue
authors.forename: Jean Claude
authors.initials: JC
authors.lastname: Pernolle
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Shioi
mesh_terms: D020418:Algal Proteins / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002477:Cells / Q000166:cytology* / Q000187:drug effects; D005452:Fluoresceins / Q000032:analysis; D005656:Fungal Proteins; D007700:Kinetics; D010838:Phytophthora / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects
chemical_list: D020418:Algal Proteins; D005452:Fluoresceins; D005656:Fungal Proteins; C061032:cryptogein protein, Phytophthora cryptogea; C018506:diacetylfluorescein