Black flat bottomed 96 well plates

Biofilms were grown in clear round-bottomed 96-well plates (SPL) to evaluate their survival after treatment, or in black flat-bottomed 96-well plates (Perkin Elmer) for measuring fluorescence. An inoculum of approximately 5x10⁷ CFU/ml was prepared in fresh medium from an overnight culture. 100 μl of this inoculum was added to the wells of a MTP. After 4 hours of adhesion the supernatant was removed and the wells were rinsed with PS. Subsequently, fresh medium was added to the wells and the MTP was further incubated for 20 hours at 37°C.

Cellomics Multiparameter Cytotoxicity 3 kit (Thermo Scientific^TM, Pittsburgh, PA, USA) was used to perform simultaneous detection of the crucial apoptotic events in SKOV-3 cells treated with artonin E. Briefly, SKOV-3 cells were seeded at a density of 5000 cells/well in a black flat-bottomed 96-well plates (PerkinElmer, Inc., Wellsley, MA, USA) and left overnight to attach. The attached cells were treated with 8 μg/mL artonin E for 24, 48, and 72 h. The treated cells were then exposed to 50 μL of live cell staining solution for 30 min. The excess medium and live cell staining solution were gently removed and the cells were then fixed with 16% paraformaldehyde. After fixation, the cells were exposed with permeabilization buffer followed by blocking buffer for 15 min at room temperature. Primary Cytochrome C antibodies and secondary DyLight 649 conjugated goat antimouse IgG were added and allowed to interact for 1 h each. Hoechst 33342 dye was used to stain nuclei in SKOV-3 cells. Stained SKOV-3 cells in the 96-well plates were analyzed using ArrayScan high-content screening (HCS) system. All experiments were conducted in triplicates and experiments were repeated twice.