ABSTRACT

HCS assays designed for microplates have been successfully transferred to the CellChip system. One of these assays-the cytoplasm-to-nucleus translocation of intracellular molecules-is a class of cell-based screens that tests the ability of candidate drug compounds to induce or inhibit transport of transcription factors from the cytoplasm the nucleus. Sensor cells arrayed on CellChips are treated with a combination of chemical entities. The assays can be run as fixed-endpoint or live-cell kinetic assays. For example, in a fixed-endpoint assay, an array of sensor cells is treated with a chemical fixative and labeled with a fluorescent nucleic acid probe and an antibody against the transcription factor or stress-associated protein labeled with another fluorescent conjugate. The test consists of measuring the fluorescence from the antibody in the nucleus (the nucleus being defined by the nucleic acid probe), versus the cytoplasm defined by the cell domain outside of the nucleus. Proprietary algorithms facilitate quantitation of the kinetics and amount of transcription factor or stress protein translocation into the nucleus over time [6]. Using a polymer-based CellChip system, we have quantified the activation-induced translocation of NF-κB, a transcription factor involved in cell stress molecular pathways, in response to tumor necrosis factor-a (TNF-a). Appropriate cellular domains on the CellChip platform were dosed with TNF-a; the cells were fixed, permeabilized, and labeled for the NF-κB p65 and nuclear domains (NF-κB Activation HitKit™; Cellomics,

Inc.). As seen in Figure 6, there was a redistribution of the transcription factor to the nucleus because of stimulation. There was up to a fourfold increase in both the normalized nuclear intensity and the normalized ratio between nuclear and cytoplasmic intensities post translocation. This increase is equivalent to the results obtained in the microplate platform [6].