title: Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro.
abstract: The aim of this study was to compare different and commonly used cell viability assays after CHO cells treatment with anticancer drug bleomycin (20 nM), high voltage (HV) electric pulses (4 pulses, 1200 V/cm, 100 µs, 1 Hz), and combination of bleomycin and HV electric pulses. Cell viability was measured using clonogenic assay, propidium iodide (PI) assay, MTT assay, and employing flow cytometry modality to precisely count cells in definite volume of the sample (flow cytometry assay). Results showed that although clonogenic cell viability drastically decreased correspondingly to 57 and 3 % after cell treatment either with HV pulses or combination of bleomycin and HV pulses (bleomycin electrotransfer), PI assay performed ~15 min after the treatments indicated nearly 100 % cell viability. MTT assay performed at 6-72 h time points after these treatments revealed that MTT cell viability is highly dependent on evaluation time point and decreased with later evaluation time points. Nevertheless, in comparison to clonogenic cell viability, MTT cell viability after bleomycin electrotransfer at all testing time points was significantly higher. Flow cytometry assay if used at later times, 2-3 days after the treatment, allowed reliable evaluation of cell viability. In overall, our results showed that in order to estimate cell viability after cell treatment with combination of the bleomycin and electroporation the most reliable method is clonogenic assay. Improper use of PI and MTT assays can lead to misinterpretation of the experimental results.
authors.affiliation: Biophysical Research Group, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos 8, 44404, Kaunas, Lithuania.
authors.forename: Baltramiejus
authors.initials: B
authors.lastname: Jakštys
authors.forename: Paulius
authors.initials: P
authors.lastname: Ruzgys
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Tamošiūnas
authors.forename: Saulius
authors.initials: S
authors.lastname: Šatkauskas
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001761:Bleomycin / Q000494:pharmacology*; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects*; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D003412:Cricetulus; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D066298:In Vitro Techniques
chemical_list: D000903:Antibiotics, Antineoplastic; D001761:Bleomycin