Matrigel coated boyden chamber inserts

To assess tumor cell migration, 21,000 cells were seeded into each chamber of sterile culture inserts (ibidi, Germany), which were placed in 6-well plates. 12 hours after seeding, cells were treated for one hour with 10 μg/ml Mitomycin C (Sigma, Germany) in the incubator and insert were removed. Pictures were taken with the Nikon Eclipse Ti microscope using the Nikon Imaging Software NIS-Elements 3.20.02 at the indicated time points and analyzed with the software tool ImageJ to quantify the kinetic of gap closure over time.
To determine tumor cell invasion, FaDu-shKLK6 and FaDu-Mock cells were treated for one hour with 10 μg/ml Mitomycin C (Sigma, Germany), trypsinized and 100,000 cells were seeded in matrigel-coated Boyden chamber inserts (BD Bioscience, Germany), which were placed in 12-well plates. After 48 hours, non-invading cells were removed using cotton swaps and remaining cells were fixed for ten minutes in 4 % PFA and stained with Hoechst 33342 (Calbiochem Merck, Germany). Following mounting with Mowiol, five representative pictures were taken with the Nikon Eclipse Ti microscope using the Nikon Imaging Software NIS-Elements 3.20.02, and the amount of invaded cells was counted with the software tool ImageJ.

50,000 cells resuspended in 350 μl of serum-free medium were seeded into the Matrigel-coated Boyden chamber inserts (BD Biosciences). In the bottom chamber 750 μl medium with or without 10% FCS served as chemoattractant or negative control, respectively. Cells were permitted to migrate for 36 hrs, then fixed and stained with crystal violet. Pictures were taken on a Zeiss Observer. A1 microscope. The value of the migrated cells was calculated from at least three wells for each experiment group and analyzed with the Photoshop CS3 extended measurement feature.