Protocol

Cancer Cell Culture

Volume 731 of the series Methods in Molecular Biology pp 285-291

Date:

Real-Time Cytotoxicity Assays

  • Donald WlodkowicAffiliated withThe BioMEMS Research Group, Department of Chemistry, University of Auckland Email author 
  • , Shannon Faley
  • , Zbigniew Darzynkiewicz
  • , Jonathan M. Cooper

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Abstract

Validation of new therapeutic targets calls for the advance in innovative assays that probe both spatial and temporal relationships in signaling networks. Cell death assays have already found a widespread use in pharmacological profiling of anticancer drugs. Such assays are, however, predominantly restricted to end point DEAD/LIVE parameter that provides only a snapshot of inherently stochastic process such as tumor cell death. Development of new methods that can offer kinetic real-time analysis would be highly advantageous for the pharmacological screening and predictive toxicology. In the present work we outline innovative protocols for the real-time analysis of tumor cell death, based on propidium iodide (PI) and SYTOX Green probes. These can be readily adapted to both flow cytometry and time-lapse fluorescence imaging. Considering vast time savings and kinetic data acquisition such assays have the potential to be applied in a number of areas including accelerated anticancer drug discovery and high-throughput screening routines.

Key words

Cytotoxicity Real-time assays Antitumor drugs Flow cytometry Time-lapse microscopy