Detecting the FRET Response of the GeneBLAzer® Cell Line D1 CRE-bla CHO-K1 to Agonists and Antagonists using Microplate Cytometry
Poster Feb 01, 2007
Christopher Lupton, Randy Hoffman, Paul Wylie and Wayne Bowen
The GeneBLAzer® CHO.K1-D1 cell line (Invitrogen) stably expresses both the ß-lactamase gene downstream of the cAMP response element (CRE) and the dopamine D1 receptor. Stimulation of the cells with dopamine D1 receptor agonists, results in transcriptional activation of the ß-lactamase gene through CRE. A FRET-enabled substrate (CCF4-AM) fluoresces green, in the absence of ß- lactamase reporter activity, and blue when cleaved. This technology has been measured by bulk fluorescence readers, which report data on a whole well basis.
In this study, the violet laser in the Acumen Explorer 405 microplate cytometer was used to excite CCF4-AM substrate and the resulting fluorescent emissions simultaneously detected in the blue and green channels.High content data for dopamine D1 receptor activation were calculated from ratios of blue to green fluorescence in cell populations. We have shown that the Acumen Explorer 405 has the ability to accurately measure ß- lactamase activity, and generates good fold activations above baseline.
We have also shown that we can obtain toxicological data from the same plate, thereby providing additional, valuable information during the screening process. When coupled with short read times of less than 10 minutes per plate, this technology provides an excellent opportunity for functional genomic applications.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE