Chemotaxis tool plugin for imagej

Cells were plated on gelatin coated 6 or 12 wells plate and cells were allowed to adhere for at least sixteen hours. Experiments were performed in a humidified chamber with 5% CO₂ at 37 °C in the presence of DMSO or SMIFH2. Cells were imaged every five minutes on a Zeiss Axio Observer Z1 microscope (Carl Zeiss) equipped with a LD Plan-Neofluar Ph2 20x (N.A. 0.40) objective, operated with Zeiss Microscope Software ZEN 2012. Individual cells were tracked using Manual Tracking plugin for ImageJ. Average distance, speed and directionality of movement were computed using the Chemotaxis Tool plugin for ImageJ provided by ibidi GmbH (http://www.ibidi.com).
Cells entering mitosis were scored manually and defined as follows: a cell entered mitosis when its flat and spread appearance changed to a round-up, yet adhesive state. Initial number of cells was counted manually in each field of observation and percentage of cells entering mitosis every hour was calculated using this initial number of cells as reference.

Cell migration was monitored by time-lapse microscopy using an Olympus FV1000 confocal microscope. Optimal growth cell conditions were maintained using on-stage incubator chamber at 37°C in an atmosphere of 5% CO₂. Z-stacks of 200 μm were acquired using 12–15 μm steps; initial and final z positions were chosen to be at least 50μm separated of the surface or the plate interface. Different areas (4 to 9 areas) were acquired per sample (each individual area of 0.0187 mm²) to cover at least 60% of the total area of the well. Image stacks were projected as XY images for migration analysis. Trackmate plugin from FIJI was used to analyze the time-lapse images using LoG (Laplacian of Gaussian) detector, assuming a blob diameter of 10 pixels (all images were 512 pixels, 2.67 μm per pixel) and threshold of 1 pixel, without sub-pixel localization. LAP tracker option was chosen allowing frame to frame linking and closing of 15 pixels in 3D migration experiments and 25 pixels in 2D migration experiments. Data was filtered to only account for cells visible during the total time of the experiment. Raw data from Trackmate was analyzed using the Chemotaxis tool plugin for ImageJ (Ibidi, Germany) to obtain total migration distance, net displacement and directionality (ratio of net displacement to total migration distance).