rapid profiling of multiple toxicity indicators using a laser scanning imager
The drug discovery industry requires continual development and production of new drugs to address a wide range of clinical conditions and increase product portfolios. A major problem for the pharmaceutical industry is the failure of promising drug candidates very late in the testing phases. Often, there are unpredicted side-effects and toxicity issues which limit or prevent a candidate molecule being taken to market. Such failures at this stage of the product development pipeline incur substantial time and cost factors to the pharmaceutical and healthcare industry. Testing regimes that can identify and exclude toxic molecules as early as possible during the screening program will lead to significant time and cost savings.
Previous studies using imaging-based high content analysis have shown the possibility of its use in toxicity screening1. Traditional image-based systems have limited throughput capability in primary toxicity screening campaigns. Here we describe a method for high content, high throughput toxicity screening in 384 well plates with a scan time of 15 minutes per plate for whole well scans which includes both data acquisition and analysis time using TTP Labtech’s acumen® hci.
Three fluorophores were used to monitor the hepatoxic effects of a small panel of commercially available compounds on HepG2 cells. The 405 nm laser-excited Hoechst 34580 stains all nuclei and facilitates enumeration of total cell number (giving a measure of cellular proliferation). Additionally, Hoechst nuclear half width measurements give a measure of nuclear condensation which occurs during apoptosis. The 488 nm laser-excited TMRM is a cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria. TOTO-3 is a 633 nm laser-excitable fluorophore which stains the nucleus of dead/dying cells where the cell membrane has been compromised, thus providing a number of total dead cells per well.