Dual Stable Cell Line Alternatives
Promega's (www.promega.com) presentation featured bioluminescent cell-based assays, specifically the dual luciferase assay it has developed. Bioluminescent cell-based assays are well-suited for high-throughput screening because of their high sensitivity, wide dynamic range, and minimal interference by the compounds. "Our goal is to make things as simple for the scientist as possible," said Erika Hawkins, senior investigator at Promega. "To that end, we constructed dual stable cell lines to reduce false positives resulting from compound-induced cytotoxicity in antagonist screens."
In agonist screening, single-reporter, uncorrected data cannot distinguish between specific down regulation and cytotoxicity, as both responses exhibit a similar decrease in luminescence. In Promega's Dual-Glo Assay, however, the use of dual reporters can discriminate these responses.
"Detection systems need to be optimized for a rapid half-life, which means decreasing the time to get a good response, and increasing the window of opportunity for a good response," Hawkins said. "Using dual stable cell lines reduces off-target response and gives us data that are easy to see and track."
Promega constructs stable dual luciferase cell lines using two plasmids. One plasmid expresses firefly luciferase genes under the control of a response element (e.g., CRE or NFAT) and a hygro-mycin selectable marker that has been stably transfected into master cell lines. The second plasmid expresses target GPCR (e.g., dopamine receptor D1 or muscarinic 3 receptor M3R) and a Renilla luciferase-neomycin selectable marker fusion. Using destabilized luciferases resulted in faster signal responses and shorter assay times and thus a general reduction in opportunity for cytotoxicity from library compoundsACEA Biosciences (www.aceabio.com), presented label-free technology and applications for cell-based assays. The core of ACEA's RT-CES system is the microelectronic cell sensor arrays integrated into the bottom of microtiter plates. "For cell-based assays, cells are grown in the individual, sensor-containing wells of the microtiter plate. The electronic sensors provide continuous, quantitative information concerning the biological status of the cells present in the well."
Changes to the biological status of the cells are measured automatically and in real time by the RT-CES system. There are no labeling steps with expensive reagents, and the entire process is noninvasive and harmless to the cells. "For cell-based assays, we are dealing with a living thing, a live cell," said Xu. "Without knowing the cell status before the treatment and without knowing the kinetics of live cell response to the exposure of chemical compounds, it will be hard to get a consistent result between experiments."
The actual variable being measured by the RT-CES system is derived from the change in electrical impedance as the living cells interact with the biocompatible microelectrode surface in the microplate well. Using a proprietary algorithm, the signal is converted to a specific parameter called the Cell Index, a measure of what the cells are actually doing over time growing, spreading, changing shape, dying, responding to specific stimuli, and so on.
A range of cell-based assays can be performed on the RT-CES system, including cell proliferation, cytotoxicity profiling, ADCE, cell adhesion, and functional analysis of receptors, such as GPCR, growth factor receptors, and death receptors.
Xu emphasized the importance of label-free capability. "This is a QC issue with cell-based assays. You need to know that your cell quality is good. I know how important it is for scientists to have the tools they need to do their work."
Zhong Zhong, vp of drug discovery technologies at Cell and Molecular Technologies (www.cmt-inc.net), demonstrated that division-arrested cryopreserved cells performed with better consistency than actively cultured cells in screening applications. These ready-to-use reagents can be thawed, plated, and used in screening to improve consistency, convenience and cost efficiency.
"Cell-based assays are becoming increasingly popular for small molecule discovery in high-throughput screening," said Zhong. "Advanced HTS technologies require standardized, consistent cell culture as reagents in order to generate reliable results. Division arrest technology, which is a scalable process, allows the use of frozen assay-ready cells for screening as if they are ordinary assay reagents like enzymes or membrane preparations."
The division-arrest technology effectively uncouples the laborious, time-consuming process of cell production from the robotic screening process and gives users the confidence to focus on assay development.