Fluorescence-based cell viability screening assays using water-soluble oxygen probes.
A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 microM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (10(5) to 10(7) cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability.
Hynes
James
J
Biochemistry Department, University College Cork, Ireland.
Floyd
Suzanne
S
Soini
Aleksi E
AE
O'Connor
Rosemary
R
Papkovsky
Dmitri B
DB
D017209:Apoptosis; D001681:Biological Assay / Q000379:methods; D015374:Biosensing Techniques / Q000379:methods*; D002470:Cell Survival; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology; D007377:Interleukin-3 / Q000096:biosynthesis; D008163:Luminescent Measurements; D008403:Mass Screening / Q000191:economics / Q000379:methods*; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007377:Interleukin-3; C085925:calcein AM; D010100:Oxygen; D004121:Dimethyl Sulfoxide