Computational protocol: Epidermal growth factor receptor function in the human urothelium

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Protocol publication

[…] For migration studies, urothelial cells were cultured on TPP tissue plate 60 (TPP Techno Plastic Products AG, Trasadingen, Switzerland) inside PC4L-0.5-CoverWell Perfusion Chambers (Grace Bio-Labs Inc, Bend, Oregon, USA) at a concentration of 100,000 cells/ml in culture medium containing 20 mM HEPES (1 M, H0087, Sigma-Aldrich), and sealed with two 22 × 22 mm coverslips and melted VALAP (1:1:1 weight of vaseline lanolin, paraffin wax). Cells were then incubated with cetuximab (1.5 μM) or PBS (control). The plate was put in a thermostatic cell incubation chamber for 1 h to allow cell attachment, after which the plate was inverted and an Axioplan 2 imaging microscope with a motorized stage (Carl Zeiss Microscopy GmbH) was used to assess migration. Four representative spots per well were selected, and the cells were photographed every 5 min for 48 h using a macro in KS 400 (version 3.0, Carl Zeiss Vision GmbH). The resulting time-lapse images were made into videos using Images to Video (version 3.0.0, Jaromir Sivic).Films were analyzed using the image analysis software ImageJ (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2014) and the cell tracking plugin MTrackJ (created by Erik Meijering; www.imagescience.org/meijering/software/mtrackj). Five cells per film were selected and tracked frame by frame in order to measure the mean velocity of these cells. The paths of the tracked cells were unknown at the time of selection, and 2–3 cells that were alone and 2–3 cells that were part of a small cell island were chosen and tracked per film. The analysis was blinded. The mean velocity of the five cells was calculated, representing the mean velocity of each well, and these values were used to calculate the mean velocity of each treatment group. An area of each film covering 150–200 cells was monitored, and the number of cells within this area was counted at 120, 520 and at 920 min after seeding. Moreover, the percentage of cells forming mitotic rounding at these time points was calculated []. […]

Pipeline specifications

Software tools ImageJ, MTrackJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Urinary Bladder Neoplasms