Tracy Worzella1, Andrew Niles1, Mike Valley1, Trista Schagat1, Jerome Lasker2, Judy Raucy2, Kevin Kopish1, Pam Guthmiller1, Terry Riss1, Jim Cali1 and Cris Cowan1
1Promega Corporation, Madison, WI, USA; 2Puracyp Incorporated, Carlsbad, CA, USA
Reporter gene assays in whole cells have found widespread use as sensitive indicators of activity for a wide variety of cellular signaling events. Inclusion of a cell viability control component in this assays is often overlooked but is pivotal for ensuring that the interpretation of data is not skewed in those cases where reporter response is affected by changes in overall cell health. Herein, we present a multiplex approach that combines a non-lytic assay to assess cell viability with a next generation reporter gene assay to monitor firefly luciferase activity. Viability is determined using a cell permeable substrate that, upon cleavage by intracellular proteases, produces a fluorescent signal that is directly proportional to viable cells with intact membranes. Reporter gene activity is then assessed in cell lysates by measuring luciferase activity.
We show that this multiplex assay can be utilized for different purposes, including transfection optimization studies to guide in optimizing DNA:lipid transfection ratios to give robust firefly luciferase expression without causing cell toxicity. Another use encompasses a novel drug-drug interaction screening system where nuclear receptor activation by new chemical entities is tested while monitoring cell health. Lastly, the utility of the viability assay is shown in those instances where global cytotoxicity impacts reporter gene expression. Not only is this assay combination multi-purpose, but the addition-only reagents are easy to use and scalable in research as well as automated screening environments.