Eclipse ti e ds qi2 microscope

To evaluate the effects of EV-CPC-N, CPC-P and CF-P cultured with EV-CM or with regular medium (control groups) were fixed in 4% paraformaldehyde for 20 min at room temperature after 3 days of culture. After blocking with 10% donkey serum, cells were incubated with primary antibody against human α-sarcomeric actin (Di Meglio et al., 2010b (link)) or fibronectin (both from Sigma-Aldrich), for 1 h at 37°C, then washed and incubated with secondary antibody conjugated with fluorescein (Jackson ImmunoResearch Europe, Newmarket, United Kingdom) at 37°C, for 1 h. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI) (Merck Millipore) and stained area of culture dishes was mounted in Vectashield (Vector Labs). Microscopic analysis was performed with Nikon Eclipse Ti-E DS-Qi2 Microscope (Nikon) and marker expression was quantified by three independent observers by Nikon Imaging Analytical Software (NIS Elements Analysis 4.50) (Nikon) and expressed as mean fluorescence intensity ± SEM.

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.

EV-CPC-N were used to prepare a conditioned medium (EV-CM), to test effects produced by EVs on both CPC-P and CF-P. EV-CPC-N were resuspended in the medium specific for each cell population and added to CPC-P and CF-P culture dishes at the final concentration of 0.1 mg/ml (Barile et al., 2014 (link)) and cultured for 48 h. During the culture, the morphology of cells was evaluated by phase contrast microscope observation with Nikon Eclipse Ti-E DS-Qi2 Microscope (Nikon Corporation, Tokyo, Japan).