Metamorph 1 image analysis software

The in vitro cell migration assays were performed in modified Boyden chambers (NeuroProbe Inc., Bethesda, MD) with porous membranes pre-coated with 10 mg/mL of fibronectin or 50 mg/mL fibrinogen for 5 h at 37 C as previously described [35] .
Cell velocity was calculated from time-lapse video-microscopy as previously described [36] . Briefly, human glioblastoma cells were trypsinized and then seeded on fibrinogen pre-coated 24well plates at low confluence (10 4 cells/cm 2 ) and allowed to adhere at 37 C for 2 h. The plates were placed on an inverted Nikon microscope equipped with a heated stage and 5% CO2 supply. Three fields per well were imaged and followed at 5 min intervals over 3 h with a Coolsnap HQ camera (Photometrics, Tucson, AZ) operated by NIS-elements AR 2.30 software (Nikon). Manual single-cell tracking was performed by using Metamorph 1 image analysis software (Roper Scientific, Evry, France). Migration tracks were used to calculate total migration distance, distance to origin, velocity and directional persistence of cell migration. The distance to origin was determined as the net translocation between the initial and the final position. Velocity was calculated as the total migration distance divided by 3 h. Directional persistence was calculated as the ratio of the distance to origin to the total distance migration.

The effect of E. elaterium seed oil on in vitro angiogenesis was examined by using the HMEC-1 cells capillary-like tube formation assay. Briefly, a 96-well plate pre-coated with 100 mL Matrigel per well was prepared and solidified for 1 h at 37 C. HMEC-1 cells were plated on Matrigel-coated wells at a density of (5.000 cells/100 mL) and incubated for 6 h at 37 C and 5% CO 2. The formation of capillary-like tubular networks was observed with a DM-IRBE microscope (Leica, Rueil-Malmaison, France) coupled with a digital camera (CCD camera coolsnap FX, Princeton Instruments, Trenton, NJ). The percentage of tubule area was quantified by image analysis using Metamorph 1 image analysis software (Roper Scientific, Evry, France) as previously described by Pasquier et al. (2004) [38] .