Thincert tissue culture inserts

The cells were allowed to grow in a culture dish overnight, and a scratch approximately 3 mm in width was created in the monolayer using a pipette tip. After washing twice with PBS, the cells were subjected or not subjected to heat shock (42°C for 30 min), and images were captured after 24 h. The migratory behavior of the hDPCs was assessed using a transwell migration assay (Chemotaxis Cell Migration Assay kit, Chemicon). hDPCs were seeded onto Thincert™ tissue culture inserts (pore size = 8 μm; Greiner Bio-One, Frickenhausen, Germany) in DMEM with 10% FBS at a density of 1.5 x 10⁵ cells per insert. The cells were subjected or not subjected to heat shock and allowed to migrate for 24 h. Under some conditions, inhibitors against Pin1 and MAPKs were also added to the hDPCs. After 24 h, the hDPCs that had transmigrated towards the lower surface of the filter were fixed with methanol, stained with hematoxylin for 5 min and washed with PBS. The cells in five random microscopic fields per well were imaged using an Olympus IX2-SLP inverted microscope (Japan) at 100X magnification. The numbers of migrated cells on the lower side of the membrane were counted in five randomly selected areas, and the values are expressed as the mean area percentage.

Cells were grown in ThinCert™ Tissue Culture Inserts (8 µm pore size, Greiner, Mount Joy, PA, USA) with a serum-free medium in the bottom plate. Treatments were similar to spontaneous migration except for FB1: the first 24 h of treatment were conducted on cells seeded in 12-well plates; cells were then trypsinized and re-seeded in the inserts for the next 24 h of treatment. To induce oriented migration after treatment, inserts were filled with a serum-free medium, and bottom chambers contained either a serum-free medium (spontaneous migration) or 250 ng/mL insulin-like growth factor-1 (IGF-1, Sigma-Aldrich) diluted in DMEM/FFA BSA (stimulated migration). After 5 h of migration at 37 °C, migrating cells were fixed for 20 min with 4% paraformaldehyde (PFA), stained for 10 min with 0.5% crystal violet diluted in 4% PFA, and washed with phosphate-buffered saline (PBS). Cells that did not migrate through the insert were wiped out with cotton swabs. Ten images per insert were taken with a wide-field fluorescence microscope, Observer Z.1 (20x objective). Using the ZEN software, the total area of migrating cells was determined and divided by the area of a single cell (mean of 10 cells for each condition) to evaluate the number of cells per area. The oriented migration was calculated by subtracting the number of spontaneous migration from the number of stimulated migrating cells.