Cellinsight nxt hcs platform

For the wound-healing migration assay, cells were seeded in 96-well plates and allowed to reach confluence. Growth media was then replaced by serum-free media containing 10 μg/ml mitomycin C (Sigma Aldrich) and incubated for 2 h. A scratch was made on a uniform layer of cells using a sterile micropipette tip. After washing with PBS, cells were incubated with varying concentrations of antibodies diluted in DMEM/20% Wnt3a-CM for up to 48 h and stained with 2 μM Calcein-AM (Sigma Aldrich). The plate was scanned by CellInsight™ NXT HCS platform (Thermo Scientific) at indicated time points. Wound areas were measured using ImageJ software and IC50 values were generated based on the relative wound area data against the control group. For invasion assay, cells were assessed in Transwell with CultureCoat 24-well high BME insert (Trevigen). Cells resuspended in serum-free media with different concentrations of antibodies were added into the upper compartment of a Transwell insert. The culture medium containing 20% Wnt3a-CM was used as a chemoattractant and placed in the lower compartment. After 24 h incubation, the cells were fixed with 4% PFA and stained with 0.1% crystal violet (Sigma Aldrich) in PBS. The invaded cell number in each Transwell was counted in five fields spanning the membrane.