title: In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.
abstract: OBJECTIVE
  For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis.
  
  
  METHODS
  A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity.
  
  
  RESULTS
  Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays.
  
  
  CONCLUSIONS
  Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments.
authors.forename: Katharina
authors.initials: K
authors.lastname: Helm
authors.forename: Marlena
authors.initials: M
authors.lastname: Beyreis
authors.forename: Christian
authors.initials: C
authors.lastname: Mayr
authors.forename: Markus
authors.initials: M
authors.lastname: Ritter
authors.forename: Martin
authors.initials: M
authors.lastname: Jakab
authors.forename: Tobias
authors.initials: T
authors.lastname: Kiesslich
authors.forename: Kristjan
authors.initials: K
authors.lastname: Plaetzer
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001681:Biological Assay; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004817:Epidermis; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007202:Indicators and Reagents / Q000737:chemistry*; D007211:Indoles / Q000494:pharmacology; D008027:Light; D009336:Necrosis / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D010078:Oxazines / Q000737:chemistry*; D014230:Triazoles / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry*
chemical_list: C103118:4,5,6,7-tetrabromo-2-azabenzimidazole; D015415:Biomarkers; D007202:Indicators and Reagents; D007211:Indoles; D010078:Oxazines; D014230:Triazoles; D014966:Xanthenes; D003609:Dactinomycin; C005843:resazurin; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; C064758:Ro 31-8220
keywords: Apoptosis; Cell death; Cytotoxicity; Necrosis; Viability analysis