Inverted phase contrast microscope

Videomicroscopy measurements were performed and analyzed as described previously [55 (link)–57 (link)]. Briefly, melanoma cells were plated in the inner 8 wells of 24-well plates (Corning Incorporated, Corning, NY) in DMEM medium supplemented with 10% FCS. The medium was changed to CO₂-independent medium (Gibco-BRL Life Technologies, Carlsbad, CA) with 10% FCS and 4mM glutamine after the overnight cell attachment. In order to reduce evaporation from the inner wells, the outer wells were filled with medium. Cells were kept in a custom designed incubator built around an inverted phase-contrast microscope (World Precision Instruments, Sarasota, FL) at 37°C and room ambient atmosphere. Images of 3 neighboring microscopic fields were taken in every 5 min for 1 day before and 2 days after the treatment with zoledronic acid, dacarbazine or both. For migration data the captured phase contrast microscope pictures were analyzed individually with a cell-tracking program enabling manual marking of individual cells and recording their position parameters into data files. The parameter migrated distance is calculated by averaging for each cell the displacement for the first 24 hour interval after treatment, in two independent experiments and 3 microscopic fields.

Video microscopy measurements were performed and analyzed as described previously [36 (link)]. Briefly, cells were seeded in 24-well plates (Corning Incorporated, USA) and incubated overnight in DMEM medium supplemented with 10% FCS. CO₂-independent culture medium (Gibco-BRL Life Technologies, UK) supplemented with 10% FCS and 4 mM glutamine was applied and the plate was transferred to the custom designed incubator built around an inverted phase-contrast microscope (World Precision Instruments, USA). The experiment was performed at 37 °C in room ambient atmosphere. Images were taken every 10 mins from three neighboring microscopic fields in each well for 72 h. Migration data was captured with a manual cell-tracking program. The parameter migrated distance is calculated by averaging for each cell the displacement for the 48–72 h period after treatment, in at least three microscopic fields.

The motility responses of human LECs to apelin were analysed by long-term time-lapse videomicroscopy in 2D cell cultures as described recently [25 (link)]. Briefly, LECs were plated in a 24-well plates (Corning Inc.) in a serum-free EBM-2 medium. The medium was changed to CO₂-independent medium (Gibco, Paisley, UK) after the overnight cell attachment and apelin-13 or apelin-13(F13A) (an APJ antagonist) [14 (link), 37 (link), 39 (link)] was added into the medium at 50 or 100 nM concentrations. Cells were kept in a custom designed incubator built around an inverted phase-contrast microscope (World Precision Instruments, Florida, USA) at 37°C and room ambient atmosphere. Images of 3 neighbouring microscopic fields were taken in every 5 min for 1 day before and 2 days after the treatment. For migration data, pictures were analysed individually with a cell-tracking program enabling manual marking of individual cells and recording their position parameters. The parameter migrated distance is calculated by averaging for each cell the displacement for the first 24 hour interval after treatment, in two independent experiments and 4 microscopic fields.