Friday, January 25, 2013

Bottom-reading Live Cell Assays

Live-cell assays are becoming an invaluable tool in the drug discovery process. When performing such assays in a low- or high-throughput screening (HTS) format, the assay is typically performed in a 96- or 384-well density microplate, respectively. In a recent poster presentation at the 2013 SLAS conference we have demonstrated the high-performance bottom reading optical system of the Synergy NEO HTS multi-mode reader using the GloSensor™ technology from Promega. GloSensor is a live-cell luminescence-based kinetic assay typically performed in a solid white microplate known to give the highest signal intensity. The ability to multiplex this assay for use with other spectroscopic methods may require the use of a microplate with different characteristics. To test feasibility of alternative microplate types the assay was performed using several plate types with a variety of reading parameters on the Synergy NEO; luminescence intensity was measured either from the top or bottom of the plate using either full light or a specially designed aperture for 1536-well density microplates.

Comparing raw luminescence counts can often provide a misleading measure of assay performance Signal-to-background is typically a more useful parameter for determining the robustness of an assay. Here we compare fold-induction, which is a ratio of treated to untreated cells, or the signal to background, for each plate type and read parameter for the GloSensor variant 20F: a relatively weak biosensor (Figure 1).

fold induction

Figure 1. Fold induction represents activation of endogenous adenylate cyclase by 10 ┬ÁM forskolin leading to increased production of cAMP. Fold induction is calculated as the ratio of wells containing either induced to uninduced cells.

It is apparent from the graph that bottom reading on the Synergy NEO can provide comparable S/B. Thus bottom reading provides sufficient S/B to allow multiplexing of the assay possibly with fluorescent methods. For further information regarding the Synergy NEO and GloSensor assay please see the GloSensor poster presentation.

By: BioTek Instruments

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