Keratinocytes were harvested and seeded (1 × 104 cells/well) on the various substrates in the wells of a 48-well culture plate (NUNC). After three days of culture in DKSFM keratinocytes were fixed with 4% paraformaldehyde/PBS for 15 min at RT, incubated in PBS/1%BSA for 1 h at RT before polymerized actin was stained with 1 unit/ml of phalloidin-Alexa 488 (Molecular Probe) and nuclei with DAPI. Using a 20x objective and an Olympus IX-81 high content screening inverted microscope (Olympus), 8 by 8 non-overlapping quadrants were imaged. Cell size as delineated by the stained actin cytoskeleton was determined using the Cell Profiler software75 (link).
Keratinocyte movement was assessed on either Fib-Mat, collagen I coating or uncoated TCP. Keratinocytes were seeded as described and following an overnight incubation to allow cell adhesion; live cell images were collected using Olympus IX-81 high content screening inverted microscope (Olympus). Time-lapse images were taken at 15-minute intervals over 2 days using a 10x objective.
Keratinocyte movement was assessed on either Fib-Mat, collagen I coating or uncoated TCP. Keratinocytes were seeded as described and following an overnight incubation to allow cell adhesion; live cell images were collected using Olympus IX-81 high content screening inverted microscope (Olympus). Time-lapse images were taken at 15-minute intervals over 2 days using a 10x objective.
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