(NTR_SBS2001.pdf - 85 Kb)

The LEADseeker Cell Analysis System is an integrated screening system combining fast confocal line scanning with optimised software enabling real time data analysis of live cell assays at subcellular resolution. It utilises a rapid autofocus approach and optics that can image samples in traditional 96 well microtitre plates. Three excitation laser lines, UV (364nm), blue (488nm) and red (635nm), are simultaneously focused to a line on the sample. Fluorescence emission from the entire line is imaged through a confocal slit mask, and spectrally separated onto three cameras operating in the blue (430-495 nm), green (505-595 nm) and red (605-760 nm) regions. Advances in instrumentation such as the LEADseeker Cell Analysis System requires the development of appropriate assay systems. This requirement has lead to the development of a new intracellular live cell fluorescent gene reporter system. The gene reporter system comprises the bacterial enzyme E.coli nitroreductase (NR) and a proprietary cell permeable quenched cyanine dye, which functions as the substrate for NR. Intracellular cleavage of the ester groups results in retention of the substrate molecule inside live cells, an essential requirement for a real time kinetic imaging system such as the LEADseeker Cell Analysis System. Addition of the substrate to a cell expressing NR restores the fluorescence of the quenched fluor permitting detection of gene reporter expression. In this poster we present data from a model assay system. A plasmid was constructed containing the NF-kB response element upstream of NR. Transient transfections were established in HeLa cells and NR expression induced with the addition of TNF-a. Data are presented describing steps involved in optimising an assay on the LEADseeker Cell Analysis System, dose dependent expression of NR and inhibition of gene expression by a neutralising antibody to TNF-a.