Accurate Cytotoxicity and Proliferation Determination: Advantages of a High-Throughput Phenotypic Approach Over ATP Luminescence Assays.

Cell viability and proliferation assays are a fundamental tool in the drug discovery process and are used to evaluate both the antiproliferative potency and toxicity of compounds. Some lead discovery groups generate cell viability data for up to two million compounds per screen, so any method used to assess these parameters needs to deliver not only on data quality but also on throughput and assay cost per well. Most methods used to determine cell viability cannot deliver on all three of these requirements, so compromises have to be made. Here we show the development and implementation of a cost-effective, no-wash phenotypic assay to simultaneously report the number of cells, percentage of live cells, and cell cycle phase distribution as markers of proliferation and viability. We demonstrate that this assay can be applied to high-density plate formats and be imaged and analyzed in 8 min per plate on a laser scanning imaging cytometer. By comparing the drug-responses of several well-characterized anticancer drugs on HeLa cells, we highlight the key differences between the phenotypic assay and a commercial ATP luminescence detection system.

Advances in laser scanning imaging cytometry for high-content screening.

The advent of high-content screening more than a decade ago remodeled drug discovery workflows by recasting the role of cell-based approaches in target identification, primary screening, lead optimization, and toxicity. The ability to identify and quantify compound effects on multiple cellular functions allows for rapid characterization of chemical libraries. Laser scanning imaging cytometry (LSIC) is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using laser scanning excitation through a specialized F-theta scan lens. This configuration results in rapid whole well scanning and large depth of field. The recent availability of systems equipped with multiple sources of laser excitation and arrays of detectors for spectral analysis has significantly increased its applicability through enabling more fluorescent reagents and higher levels of multiplexing. LSIC is being used most extensively for phenotypic screening especially in areas such as cell health, RNA interference (RNAi) screening, and three-dimensional cell models. This review communicates advances in LSIC and how it is being applied by presenting an overview of the technology and a range of real-world case studies.

Subunit dimers of alpha-hemolysin expand the engineering toolbox for protein nanopores.

Staphylococcal α-hemolysin (αHL) forms a heptameric pore that features a 14-stranded transmembrane ß-barrel. We attempted to force the αHL pore to adopt novel stoichiometries by oligomerizing subunit dimers generated by in vitro transcription and translation of a tandem gene. However, in vitro transcription and translation also produced truncated proteins, monomers, that were preferentially incorporated into oligomers. These oligomers were shown to be functional heptamers by single-channel recording and had a similar mobility to wild-type heptamers in SDS-polyacrylamide gels. Purified full-length subunit dimers were then prepared by using His-tagged protein. Again, single-channel recording showed that oligomers made from these dimers are functional heptamers, implying that one or more subunits are excluded from the central pore. Therefore, the αHL pore resists all structures except those that possess seven subunits immediately surrounding the central axis. Although we were not able to change the stoichiometry of the central pore of αHL by the concatenation of subunits, we extended our findings to prepare pores containing one subunit dimer and five monomers and purified them by SDS-PAGE. Two half-chelating ligands were then installed at adjacent sites, one on each subunit of the dimer. Single-channel recording showed that pores formed from this construct formed complexes with divalent metal ions in a similar fashion to pores containing two half-chelating ligands on the same subunit, confirming that the oligomers had assembled with seven subunits around the central lumen. The ability to incorporate subunit dimers into αHL pores increases the range of structures that can be obtained from engineered protein nanopores.