Cellsense entry 1

Human embryonic lungs were obtained from terminations of pregnancy donated to the Body Donation Service of the Department of Human Anatomy and Embryology of the School of Medicine of the University of Barcelona for morphological and molecular studies. Lungs were dissected from embryos in the pseudoglandular stage of development (weeks E9, E10 and E11). The lungs were treated with dispase II (Sigma-Aldrich, St. Louis, MO, USA) and the lung mesenchyme was separated from the bronchial tree. Cell images were taken by IX53 inverted microscope using cellSense Entry 1.7 software (Olympus, Center Valley, PA, USA). Mesenchymal cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in Dulbecco’s Phosphate Buffered Saline (DPBS 1x) (Invitrogen, Carlsbad, CA, USA) and immunostained using monoclonal mouse anti-vimentin clone V9 ready-to-use and monoclonal mouse anti-human e-cadherin clone NCH-38 ready-to-use (Dako Denmark A/S, Glostrup, Denmark) in order to confirm the mesenchymal characteristics of the cells.

Cell migration was measured by in vitro scratch assay [37] (link). 5*10⁵ cells were plated in a 12-well plate one day before transfection with pre-miRNAs. Twenty-four hours after transfection, the cell monolayer was scraped in a straight line to create a “scratch” with a p100 pipet tip. The migration distance (µm) was assessed at 36 h (HCC-44) or at 48 h (H23 and A-549) after transfection using cellSense Entry 1.7 software (Olympus).