Af6000 time lapse microscope

Osteosarcoma cells KHOS, MNNG/HOS, MG-63 and Saos-2 were seeded in 24 well plates (0.15 × 10⁶) and osteoblast MC3T3-E1 at (0.1 × 10⁶). Once the cells reached confluency, the monolayer was wounded with a fine pipette tip and cells were treated with vehicle or exogenous Sema3A (300 ng/ml) for 16 hours. Migration of cells was visualized on a Leica AF6000 Time Lapse microscope. Percentage of wound closure was determined using TScratch. For random migration, KHOS osteosarcoma cells were plated in 24 well plates (1 × 10³ cells/well). Cells were treated with vehicle or Sema3A (300 ng/ml). Migration was monitored for 8 hours with a Leica AF6000 Time Lapse microscope. Accumulated distance (total track length) and velocity were measured using the Chemotaxis and Migration tool in ImageJ.

Twenty-four hours after treatments, as indicated, cells were imaged using a Leica AF6000 Time Lapse microscope at 37 °C maintaining 5% CO₂ concentration. Phase contrast images were taken using a 20× phase contrast objective every five minutes for 12 h. A minimum of five positions were imaged per well with images being focussed on mitotic cells. Images were then put in sequence, producing a time-lapse video using ImageJ. Time through mitosis was defined as the time of nuclear envelope breakdown to the end of cytokinesis. Cellular death during mitosis was counted. Aberrant mitoses were defined as mitoses, which resulted in <2 or >2 daughter cells or in which spindle defects (asymmetrical, multiple poles) or lagging chromosomes were seen. A minimum of 50 mitoses were counted per condition.

Wounds were created on confluent EC monolayers using a pipette tip and migration of EC into the wounded area was visualised using time-lapse confocal microscopy (Leica AF6000 Timelapse microscope) as described^{24 (link)}. Images were captured at 3 min intervals for 20 h after wounding. The distance migrated was determined by measuring the position of the monolayer edge in relation to the starting position using LAS_AF Software.