Oris cell migration assay

HT1080 fibrosarcoma cells (ATCC# CCL-121) were cultured to 80% confluence in growth medium (MEM with 2 mM L-glutamine, 10% FBS, 1:100 non-essential amino acids, 1 mM sodium pyruvate, 1% penicillin-streptomycin and 1% gentamicin). Cells were plated in Oris Cell Migration Assay (Platypus Technologies) plates (10⁵ cells per well) following the manufacturer’s protocol. The next day, the growth medium was removed and the wells were rinsed with Dulbecco’s PBS (D-PBS; Ca⁺⁺- and Mg⁺⁺-free), and the cells were incubated in assay medium (MEM with 2 mM L-glutamine, 10% heat shocked FBS, 1:100 non-essential amino acids, 1 mM sodium pyruvate, 1% penicillin-streptomycin and 1% gentamicin) in the presence or absence of inhibitor for 20–24 h at 37°C and 5% CO₂. After incubation the cells were rinsed with D-PBS (containing calcium and 20 mM HEPES), and incubated with the Live / Dead Cell Stain Kit containing 2 µM calcein AM and 4 µM ethidium homodimer (EthD-1) for 30 min at 37°C and 5% CO₂. The fluorescence was measured at 485/528 nm excitation/emission for calcein AM, and 530/645 nm for EthD-1. The experiments were repeated independently 2–3 times for each inhibitor. The results are presented as the percentage of survival ± SD for the mean of all assays.