Solutions were decanted from the wells and fixing solution was added (100 μl/well), followed by 15-min incubation at room temperature. Fixing solution was decanted from all wells, and the cells were washed with PBS (200 μl/well). Finally, Hoechst nuclear dye (2.5 μM) in PBS was added (100 μl/well), and the wells were incubated for 20 min at room temperature (the plates can be stored at 2–8 °C at this stage if imaging is not performed immediately).
Plates were imaged on IN Cell Analyzer 1000 using 620/60-nm excitation and 700/75-nm emission filters (CytoCy5S). Images were analyzed using the Object Intensity Analysis Module. Plates were read on a Tecan Ultra using 610/20-nm excitation and 680/30-nm emission filters (CytoCy5S).
†Time-controlled dispensing of compound solutions to the cells can be avoided by completing the assay using live cells and then fixing the cells prior to imaging. Cells should be fixed at the peak induction time point. Fixing of cells reduces the handling requirements for post-assay material to BSL-1 categorization.
Assay protocol (kinetic live-cell)
Test compound (micromolar) was prepared from stocks in assay buffer, medium was removed from the cells, and the cells were washed with PBS. Test wells were treated with 90 μl of forskolin in assay medium; 90 μl of medium alone was added to control wells. Plates were incubated at 37 ºC for 5 h, after which 10-μM CytoCy5S substrate in assay medium (10 μl) was added to give a final concentration of 1 μM. The solution was incubated for an additional 3 h at 37 ºC followed by reading on a Tecan Ultra.
Note: At all stages of the live-cell ass