title: A Protocol for a High-Throughput Multiplex Cell Viability Assay.
abstract: High-throughput cell viability assays are broadly used in RNAi and small molecule screening experiments to identify compounds that selectively kill cancer cells or as counter screens to exclude the compounds that have a generic effect on cell growth. While there are several assaying techniques available, cellular fitness is often assessed on the basis of one single and often rather indirect physiological indicator. This can lead to inconsistencies and poor correspondence between cell viability screening experiments, conducted under comparable conditions but with different viability indicators. Multiplexing, i.e., the combination of different individual assaying techniques in one experiment and subsequent comparative analysis of multiparametric data can decrease inter-assay variability and increase dataset concordance. Here, we describe a protocol for a multiplexing approach for high-throughput cell viability screening to address the issues encountered in the classical strategy using a single fitness indicator described above. The method combines a biochemical, luminescence-based approach and two fluorescence-based assay types. The biochemical method assesses cellular fitness by quantifying intracellular ATP concentration. Calcein labeling reflects cell fitness through membrane integrity and indirect measurement of ATP-dependent enzymatic esterase activity. Hoechst DNA stain correlates cell fitness with cellular DNA content. The presented multiplexing approach is suitable for low, medium and high-throughput screening and has the potential to decrease inter-assay variability and increase dataset concordance as well as reproducibility of experimental results.
authors: Institute 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.|Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany. daniel.gilbert@fau.de.|Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany. daniel.gilbert@fau.de.
authors: Daniel F
authors: DF
authors: Gilbert
authors: Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany.|Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
authors: Michael
authors: M
authors: Boutros
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D002470:Cell Survival; D003584:Cytological Techniques / Q000379:methods*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D034741:RNA, Small Interfering; D014162:Transfection / Q000379:methods
chemical_list: D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate
keywords: Cell fitness; Cell viability; Cell-based assays; Drug discovery; High-throughput screening; In vitro toxicity screening; Multiplexing; RNAi screening; Target validation