Measuring Cell Invasion Using Matrigel

Cell invasion was measured using 24-well invasion chambers (BD Biosciences; San Jose, CA) with the Matrigel-coated film insert (8 μm pore size) as described previously [7 (link)]. In brief, A2058 cells (5 × 10⁴), which were resuspended in serum-free DMEM supplemented with 0.1% BSA, were added to the top compartment of the invasion chamber. The various compounds were preincubated in serum-free DMEM/0.1% BSA with recombinant ATX for 30 min at 37°C, followed by the addition of 1 μM LPC 18:1 to the bottom chamber. The invasion chambers were incubated at 37°C in 5% CO₂ for 16 h. Next, the filter inserts were removed from the wells and transferred to a new 24-well plate containing 4 μg/ml of calcein AM (Molecular Probes, Invitrogen) in Hank’s balanced salt solution. After a 1-h incubation, the fluorescence of invaded cells was measured with a FLEXStation 3 plate reader at excitation and emission wavelengths of 485 and 530 nm, respectively.