Cell Migration Assay for CPC-P

To evaluate the speed of migration of CPC-P cultured with EV-CM and of CPC-P of control group, scratch wound assay was performed as previously described (Castaldo et al., 2013 (link)). Briefly, cells were grown to confluence and a thin scratch was produced in straight line on culture plates with a 10 µl sterile pipette tip, leaving a cell-free zone. Cell culture dishes were first washed with medium to remove debris and then fresh medium was pipetted in the dishes. Plates were placed under Nikon Eclipse Ti-E DS-Qi2 Microscope (Nikon) equipped with stage incubator (Okolab, Pozzuoli, Italy) and the migration was documented acquiring pictures every 10 min for 8 h by digital camera (Nikon). Data were analyzed by NIS Elements software (Nikon) and expressed as mean speed of migration ± SEM.