title: Determination of dynamic doxorubicin-EC50 value in an automated high-content workstation for cellular assays.
abstract: To overcome the problems of endpoint tests routinely required for EC50 determination, we utilized a novel automated high-content workstation and calculated a time-resolved EC50 value of MCF-7 mamma carcinoma cells treated with a pharmacologic agent. Measuring parameters were the cellular oxygen consumption and the extracellular acidification. These parameters were detected in real-time and label free with optochemical sensor spots in a modified 24-well sensor plate. In particular, the objective was to compare the measuring data of the workstation with a classical standard resazurin cell assay and to transfer the benefit of continuously recorded metabolic data of the workstation to practical time-resolved information about the effect of the applied active reagent (doxorubicin). MCF-7 cells were treated with a broad range of doxorubicin concentrations (100 μM to 1 nM) over 24 h and cellular activities were investigated with both, the resazurin assay and the automated workstation. Twenty-four hours after treatment, the resazurin assay showed an EC50 value (6.3 μM) which was about one decade above the value obtained from oxygen consumption rate (0.37 μM) and extracellular acidification rate (0.72 μM), measured with the workstation. Presumably, the differences are due to the different metabolic nature and regulation behind these measuring parameters. By polynomial fitting of continuously recorded metabolic data, we were able to point out a dynamic, time-resolved EC50 characteristic for different time points. The workstation is a powerful tool to record in vitro kinetic data of pharmacologic effects in vital cells in an automated experimental run.
authors.affiliation: Heinz Nixdorf-Chair for Medical Electronics at the Technical University of Munich, Theresienstr. 90, Gebäude N3, 80333 Munich, Germany. peter.wolf@tum.de
authors.forename: Peter
authors.initials: P
authors.lastname: Wolf
authors.forename: Florian
authors.initials: F
authors.lastname: Hartl
authors.forename: Martin
authors.initials: M
authors.lastname: Brischwein
authors.forename: Bernhard
authors.initials: B
authors.lastname: Wolf
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D010078:Oxazines; D010101:Oxygen Consumption; D014966:Xanthenes
chemical_list: D000903:Antibiotics, Antineoplastic; D007202:Indicators and Reagents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D004317:Doxorubicin