Incucyte zoom

To assess apoptosis induction and proliferation, 5000 cells were seeded per well in BD Falcon black tissue culture treated 96 well plates (SLS).
After 24 h media replaced to fresh DMEM containing 25 mM or 5 mM glucose and the IncuCyte Kinetic Caspase-3/7 Apoptosis Assay Reagent (Essen BioScience) was added. Apoptotic cells can be detected by a fluorescence signal if caspase 3/7 have been activated. Cells were imaged using an IncuCyte ZOOM (Essen BioScience) under 20× lense and kept at 37°C in a humidified 5% CO2 incubator. Phase contrast and fluorescence images with two to four images per well were taken every 20 min and IncuCyte ZOOM software was utilised in real-time to measure confluency, as a proxy for proliferation, and apoptosis, respectively. IncuCyte ZOOM data and timing data were imported into Prism 6 (GraphPad) for statistical analysis and presentation.

Nuclight red transduced A549 and A549.R2 cells were seeded at a concentration of 500 cells/well in a 384-well plate and allowed to settle overnight. A 7-point titration of idasanutlin (0-50 µmol/L, Tocris, DMSO), APR-246 (0-50 µmol/L, Tocris, DMSO), cisplatin (0-20 µmol/L, Tocris, 0.3%Tween20 in 0.9%NaCl) and 75 nM cytotox green reagent (Sartorius, DMSO) was added using the Tecan D300e digital drug dispenser. After 96 hours the plate was scanned with the IncuCyte ZOOM and % survival was determined with the formula:
and % cell death with the formula:
Dose response curves were plotted with GraphPad Prism software.

MDA-MB-231 cells were seeded in low serum (2%) DMEM at a density of 2.5 × 10⁴ cells per well (100 µL/well) in 96-well IncuCyte^® ImageLock plates (Sartorius, Göttingen, Germany) and allowed to adhere overnight. The following day, the center of each well was scratched using the WoundMaker Tool (Sartorius) and the wounded monolayer washed with low serum medium to remove non-adherent cells before adding inhibitors (100 µL/well). Wound healing images were acquired by real-time imaging using the IncuCyte Zoom live cell analysis system every 6 h for 24 h. Relative wound density was analyzed using IncuCyte Zoom and graphed using GraphPad Prism.