Scratch Wound Healing Assay Using IncuCyte Live Cell Imaging System

CHO-K1 cells transfected with EGFP, Y2R-EGFP, and Y5R-EGFP were seeded in IncuCyte® ImageLock 96-well plates at a density of 2–2.5 × 10⁵ cells per well. Eighteen hours after seeding, a scratch was made in the confluent monolayer using the 96-well Wound Maker™. Cells were then washed with serum-free medium to clear any floating cells within the scratch prior to treatment. The subsequent migration monitoring was performed in media with three different serum concentrations, 10%, 1%, or 0.1% FBS, depending on the experimental design. For the low-serum conditions, cells were primed in serum-free media for 6 h before creating the scratch and treating with media supplemented with 1% or 0.1% FBS and NPY or its receptor antagonists, when desired. Subsequently, the 96-well-plates were placed in the IncuCyte live cell imaging system (Sartorius, Goettingen, Germany) and the images collected every 2 h during the incubation time. The IncuCyte ZOOM software generated a wound width (WW) migration metric, which was used to calculate the migration distance (MD) according to the following formula: MD = (WW_T0 – WW_Tn)/2.