Incucyte

EdU cell proliferation kit (Thermo Fisher) was used according to manufacturer protocol. For WNT inhibition experiments, cells were treated without or with 10uM WNT-C59 (Selleckchem) for 48 h prior to EdU treatment. For WNT activation experiments, cells were treated without or with 3uM CHIR99021 (Tocris) for 24 h prior to EdU treatment. Cells were then treated with 10uM EdU for 1 h, fixed and undergone Click-iT chemistry to stain for EdU in cells. EdU-treated cells are analyzed through the BD FACS Canto II machine using the BD FACSDiva Software (BD Biosciences).
For growth assays by Incucyte (Sartorius), phase objective confluence is calculated by Incucyte after imaging every 6 h over a total of 60 h. 150,000 cells per well are plated in a 12 well plate. 16 images are taken in different sections of each well to calculate the average confluence.

The cells were seeded at 1.0×10⁴ cells/well in a matrigel coated (1:20) 24-well plate and were left to adhere for 24 hours. Hereafter DMSO (control) and AZD8055 were added at the indicated concentrations in duplicate. The 24-well plates were placed in an IncuCyte (Essen BioScience, MI, USA) at 37°C in a humid 95% air/5% CO2 chamber. Three phase contrast images/well were collected with a 10× objective every 4 hours for up to 9 days after addition of AZD8055. The percentage of the area of the well occupied by cells (confluence) was calculated using software of the IncuCyte live-cell imaging system (Essen BioScience).

Cell confluency was determined using the live imaging system IncuCyte (Essen BioScience, Ann Arbor, Michigan, USA). To this end, cells were seeded and treated as described above, placed in the IncuCyte system inside the incubator, and followed for the entire incubation period. Every 3 hours the system acquired an image and confluency was measured using the algorithm in the IncuCyte software (Essen BioScience). Results are expressed as % confluency.
The crystal violet assay was performed as described previously [33 (link)]. In brief, cells were seeded and treated as described above. After 5 days of incubation the cells were fixed using 6% glutaraldehyde (Sigma-Aldrich) and 0.5% crystal violet (Sigma-Aldrich). The staining solution was removed and the plates were washed and dried overnight. The crystal violet staining was dissolved in 10% acetic acid and absorption was measured at 595 nm.

Cell invasion rates were established using our previously published method
[18 (link),19 (link)]. Using a real-time cell imaging system (IncuCyte™ (Essen BioScience, Michigan, USA). In brief, 96-well plates were coated with a thin layer of collagen by transferring 300 μg/ml of collagen type I (Life Technologies™, Carlsbad, CA) and incubating at 37°C for 30 min. OVCAR-3 and SKOV-3 (2 × 10⁴ cells per well) were grown to confluence in complete growth media. Cell-free zones were created by generating a wound with a 96-Well WoundMaker™ (Essen BioScience, Michigan, USA). The cells were overlaid with 3 mg/ml collagen type I (Life Technologies™, Carlsbad, CA) and incubated at 37°C for 30 min to create a 3D matrix. Complete growth media was added on top of the layer of collagen. Cells were imaged automatically every 3 h over a time period of 48 h. The images were processed by the IncuCyte™ software package (Essen BioScience, Michigan, USA) to measure cell invasion by obtaining the Relative Wound Density (RWD, as defined by custom algorithms within the IncuCyte™ software package). These users informed algorithms identify the wound region and provide visual representations of the segmentation parameters. Image collection was created using three to five representative phase contrast images.

Cells were assessed in wound healing scratch assays using the IncuCyte (Essen Bioscience). Cell migration assays were achieved by growing MCF7 and MDA-MB-231 cells to confluence in 96-well plates (Essen BioScience; cat. no. 4379). Wounds were made using the 96-pin wound-making tool (WoundMaker; Essen Bioscience) to simultaneously create a precise and reproducible wound in each well, 1 h after the plate was washed twice with PBS, and incubated with DMEM containing 1% FBS, 1% penicillin-streptomycin (Gibco), 4 mM L-glutamine (Gibco), sodium pyruvate (1 mM) and 0.01 mg/mL bovine insulin (Sigma). Cell migration was monitored in real time by IncuCyte, and wound width was measured by the IncuCyte software Zoom (version 2014a, Essen Bioscience, Ann Arbor, MI, USA).