Incucyte 2011a flr

Scratch wound assay was performed using the IncuCyte 2011A FLR (Essen Bioscience). Briefly, MCF10A and MCF10A CDH1-/- cells were seeded in six replicates at densities of 2.5 × 10⁴ and 3.5 × 10⁴ cells per well, respectively, in 96 well Essen ImageLock Plate (Essen Bioscience) with different coating surfaces: no coating for the uncoated condition, 2 μg/ml collagen, 2 μg/ml fibronectin, 8 μg/ml vitronectin, 8 μg/ml laminin. Cells were incubated at 37°C and 5% CO₂ and grown to 100% confluency. The usage of the Essen ImageLock Plates ensures wounds are automatically located and registered by the IncuCyte software and analyzed using wound confluence metrics. Precise and reproducible wounds were generated using the 96 PTFE pin WoundMaker (Essen Bioscience) on the confluent monolayer and cells returned to the incubator where images of cells were acquired every 1 h for 35 h under phase contrast microscopy. Wound confluence was graphed over time to quantitatively evaluate the characteristic of wound closing using the IncuCyte software, Wound Confluence v1.5.

MCF10A and MCF10A CDH1-/- cells were seeded at densities of 2.0 × 10³ and 4.0 × 10³ in three replicates in 96 well E-plates and incubated at 37°C in 5% CO₂. The growth rate was monitored in real time at 15 min intervals for 96 h using the xCELLigence platform (Roche). Both cell lines were also seeded at the same densities into 96 well flat clear bottom black plates (Corning) and grown at 37°C in 5% CO₂ and imaged every 2 h for 96 h using the IncuCyte 2011A FLR (Essen Bioscience). Confluency was determined using the IncuCyte software Confluence v1.5.