Nunc polycarbonate cell culture inserts in multi well plates

Primary hASMCs and hAECs were added to transwell chambers with 8 µm pore size (Nunc Polycarbonate Cell Culture Inserts in Multi-Well Plates; Thermo Fisher Scientific). After 6, 12, and 24 h, cells on the upper surface were removed using cotton swabs, and the cell on the bottom side of the membranes were fixed and stained with 0.1% crystal violet (Sigma-Aldrich). Migrated cells were counted in 6 randomly selected microscopic fields (×10) using the ImageJ software.

Ten-thousand cells from monoculture were seeded onto Nunc™ Polycarbonate Cell Culture Inserts in Multi-Well Plates (ThermoFisher Scientific, MA, USA; cat# 140629) in FBS-free growth medium 24 h after transfection or at the moment of seeding (ESTDAB and primary cells). Growth medium supplemented with 10% FBS was used in the bottom chamber as attractant. Cells were incubated at 37 °C for 24 h and then fixed and stained with a 0.05% (w/v) Crystal Violet solution in 20% MeOH. Cells on the top section of the insert were gently removed with a cotton swab. Four different fields were imaged for each sample with an IX71 inverted microscope (Olympus, Tokyo, JA) provided with a CellSens Imaging Software 2 (Olympus, Tokyo, JA; RRID:SCR_016238) and counted with ImageJ version 1.52.n (University of Wisconsin, WI, USA; RRID:SCR_003070). Data are showed as average count.