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A Protocol for a High-Throughput Multiplex Cell Viability Assay.

Daniel F Gilbert1,2,3, Michael Boutros4,5

  • 1Institute of Medical Biotechnology and Erlangen Graduate School in Advanced Optical Technologies (SAOT), Friedrich-Alexander Universität Erlangen-Nürnberg, Paul-Gordan-Str.3, 91052, Erlangen, Germany. daniel.gilbert@fau.de.

Methods in Molecular Biology (Clifton, N.J.)
|September 2, 2016
PubMed
Summary
This summary is machine-generated.

Multiplexing cell viability assays combine multiple indicators for more reliable screening. This approach enhances data consistency and reproducibility in high-throughput drug discovery and RNAi experiments.

Keywords:
Cell fitnessCell viabilityCell-based assaysDrug discoveryHigh-throughput screeningIn vitro toxicity screeningMultiplexingRNAi screeningTarget validation

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Assay Development

Background:

  • High-throughput cell viability assays are crucial for drug discovery and RNAi screening.
  • Current assays often rely on single, indirect indicators, leading to inconsistencies.
  • Multiplexing offers a solution to improve assay reliability and data concordance.

Purpose of the Study:

  • To develop and describe a multiplexing protocol for high-throughput cell viability screening.
  • To address limitations of single-indicator assays in identifying selective cancer cell-killing compounds.
  • To enhance dataset concordance and reduce inter-assay variability.

Main Methods:

  • Combined biochemical (ATP quantification) and fluorescence-based assays (Calcein, Hoechst).
  • Assessed cellular fitness using intracellular ATP concentration, membrane integrity, and DNA content.
  • Developed a protocol for multiparametric data analysis.

Main Results:

  • The multiplexing approach integrates ATP levels, membrane integrity, and DNA content.
  • Demonstrated potential to decrease inter-assay variability.
  • Showcased increased dataset concordance and experimental reproducibility.

Conclusions:

  • Multiplexing cell viability assays provide a more robust and reproducible screening strategy.
  • This method is suitable for various throughput levels (low, medium, high).
  • Enhances the reliability of identifying compounds with specific effects on cancer cells.