Imagexpress micro xl widefield high content screening system

Cells were trypsinized, counted and seeded at 30,000 (HT1080) or 7,500 (MEFs) cells per well in 12-well plates pre-coated with fibronectin (5 μg/ml). Plates were incubated for 45 min before fixing with 3.7% formaldehyde (15 min), washing and staining with 0.1% Crystal Violet (30 min) at room temperature. Experiments were seeded in duplicate or triplicate and at least 5 fields of view were imaged per genotype. Cell area was manually quantified using ImageJ. For HT1080 cells, areas greater than 20,000 and less than 1000 were removed from the analysis. For primary MEF experiments, cell areas greater than 20,000 and less than 500 were removed from the analysis. Significance was analysed using one-way ANOVA plus Tukey’s multiple comparison’s test.
Single Cell Motility Assay. HT1080 cells were seeded at 6000 cells per well in ViewPlate-96 Black plates (Perkin Elmer# 6005182) in triplicate or quadruplicate. The next day, cells were labeled with 0.4 μM CellTracker™ Orange CMRA Dye (Invitrogen) for 30 min to allow better cell visualization. Cells were imaged on ImageXpress Micro XL Widefield High Content Screening System (Molecular Devices). 4 sites per well were imaged every 10 min for 80 time points. Motility analysis was performed using multidimensional motion analysis within MetaXpress software. Significance was analysed using one-way ANOVA plus Tukey’s multiple comparison’s test.

The zebrafish FUCCI cell line used in this study has been previously described (Downey et al., 2011 (link)). FUCCI cells were mixed with nonfluorescent PAC2 cells at a ratio between 1:25 and 1:50 to allow tracking of individual cells in a confluent cell culture. These mixed cultures were incubated in a water bath at 28 °C for approximately 96 h under an entraining T-cycle. Cells were then imaged on an ImageXpress Micro XL Widefield High Content Screening System (Molecular Devices, Sunnyvale, CA, USA) every 40 min for 72 h. Temperature and LD cycle were maintained during time-lapse recording. Single cell tracking was performed manually in ImageJ.