Incucyte woundmaker

To investigate the wound healing efficacy of a hydrogel comprising LMWP-GFs, QCN-NE, and OXY-PFOB-NE, an in vitro scratch-wound recovery assay was performed. A total of 2.5×10⁴ HaCaT or 4×10⁴ CCD-986sk cells (in 200 µL DMEM with 10% [v/v] FBS and 1% [w/v] penicillin/streptomycin) was added to each well of a collagen-coated Essen ImageLock 96-well plate (Essen Bioscience, Ann Arbor, MI, USA), and incubated at 37°C for 48 h to produce confluent monolayers. Cell-free scratches 700–800-µm wide were created in the monolayers using a 96-pin IncuCyte wound-maker (Essen BioScience), and the detached cells were washed twice with PBS (pH 7.4). The HaCaT and CCD-986sk cells were further treated with 200 µL of hydrogel diluted with DMEM containing 0.5% FBS to apply LMWP-GFs (LMWP-EGF [500 ng/mL], LMWP-IGF-I [500 ng/mL], LMWP-PDGF-A [10 ng/mL], and LMWP-bFGF [10 ng/mL]), QCN-NE (equivalent to 0.1 µg/mL of QCN), and 30 µg/mL of OXY-PFOB-NE alone or combined. The plates with the HaCaT and CCD-986sk cells were incubated at 37°C in an IncuCyte FLR microscope (Essen Bioscience) for 12 h and 48 h, respectively, and wound recovery was quantified using IncuCyte FLR image analysis software, which detects cell edges and generates an overlay mask to calculate relative wound density.