Rtca sp system

This assay is a real-time technique to dynamically monitor cell viability. Microelectrode-coupled microtiter plates were used to culture cells, and the cell number, cell morphology, and degree of cell adhesion were reflected by the electrode impedance. Untreated cells attach to the bottom of the well and cause an increase of the cell index (CI) value due to the increased impedance. When treated with toxic compounds, the cells die and detach from the bottom, displaying a decrease of the CI value. 50 μl of culture medium was added to each well of the E-plate 96 (Roche) for background measurements. NIH 3T3 cells were then added at a concentration of 5000 cells per well for 24 hr incubation, followed by adding PG-M and morphine sulphate diluted with DMEM to contain morphine-free base concentrations of 2.6 to 168 μg/ml. Untreated cells were used as controls. The E-plate was incubated and monitored on the RTCA SP system (Roche) for 48 hr at intervals of 15 min for 48 hr. The normalized CI was calculated by CI _original/CI _{normalize time} using the RTCA software version 1.2.1. Four replicates were measured for each sample and the means are reported.