Ge in cell analyzer 2000 high content cellular analysis system

The human A549 cell line expressing Nrf2 linked to enhanced green fluorescent protein (Nrf2-eGFP), generated in our laboratory, was added to 96-well assay plates (black plates with clear bottom), then the cells were transfected with hTim3-pcDNA3.1 or vector (pcDNA3.1) for 48 h, after which 20 μM menadion (vitamin K3), an Nrf2 nuclear translocation stimulator, was added for 6 h, then the cells were fixed with 4% paraformaldehyde and the nucleus labeled with Hoechst 33342. The cells were then imaged and analyzed using a GE IN Cell Analyzer 2000 High-Content Cellular Analysis System (GE Healthcare Bio-Sciences Corp.). To measure the activation of Nrf2, the nucleus/cytosol eGFP signal intensity ratio was calculated.

Cell nuclei were stained at 37°C for 10 min with Hoechst 33432 (Sigma–Aldrich, MO, USA, 0.5 μg/ml in PBS) and propidium iodide (PI, Sigma–Aldrich, MO, USA, 10 μg/ml in DMSO) [47 (link)]. The signals of PI and Hoechst 33432 were captured and analyzed by the GE IN Cell Analyzer 2000 High-Content Cellular Analysis System (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) to evaluate the ratios of dead cells versus total cells.