Secondary screening of hits from a small molecule screening campaign typically involves performing a dose response curve to determine the potency of the hit. Often these screens are performed with cell-based functional assays. We have recently described an IL-6 secretion assay using HTRF detection technology and the ovarian cancer cell line, SKOV-3. The assay was followed up with cell viability testing using LIVE/DEAD fluorescent probes and fluorescence microscopy. The EGFR inhibitor, AG 1478 was demonstrated to inhibit IL-6 secretion in these cells and a full dose-response curve was generated for the compound which illustrated that the compound had a high potency with an IC50 of about 15 nM. Subsequent LIVE/DEAD analysis illustrated that the compound's apparent potency was augmented by cell toxicity which would similarly be indicative of a reduction in HTRF signal.
These measurements are made possible in the same microplate reader for the first time. The Cytation 3 Cell Imaging Multi-Mode Reader uses PMT-based whole well intensity measurements using spectral filters and dichroic mirrors to provide high sensitivity HTRF detection coupled with automated digital wide-field fluorescence microscopy to provide LIVE/DEAD cell viability determinations. To learn more about the capabilities of the Cytation 3, please visit our website.
By: BioTek Instruments, Peter Banks, Ph. D., Scientific Director