Imagexpress micro xls imaging system

ASC-CM was used as a “supplement” for the Keratinocyte Growth Media (KC-GM) and dosed at a 2:1 ratio based on initial volume of ASC-CM (i.e., 20-mL of ASC-CM was concentrated to 200-μl and added to 10-mL of KC-GM). KCs were plated in 2D and allowed to acclimate and achieved desired confluences (> 24 hr). KC-GM was then removed, cells were washed, ASC-CM was applied for 24-hr, and experimental assays for metabolic, mitochondrial, proliferative, or migratory activity were then performed per the manufacturer’s instructions. Metabolic activity was assessed via PrestoBlue (as previously described, n = 4), proliferation was assessed via PicoGreen (as previously described, n = 4), mitochondrial activity was assessed via MitoTracker (as previously described, n = 4), and Migratory activity was assessed via a scratch “wound” assay. KC scratch assays were performed to evaluate changes in wound size/area as a surrogate measurement of KC migration after inflicted a scratch within a confluent monolayer of KCs (n = 4). Migration images were taken using an ImageXpress Micro XLS Imaging System (Molecular Devices) and the percent of wound area closed at 24-hr was determined via ImageJ analysis).