Keratinocyte Proliferation Assay Using Ki67

Briefly, keratinocytes were harvested and then seeded as described above and at day 3, keratinocytes were fixed with 4% paraformaldehyde for 15 min at RT, permeabilized with cold 0.1% Triton X-100/PBS for 3 min and then incubated in PBS/1% BSA for 1 h at RT. The keratinocytes were blocked with 10% goat serum/1% BSA/PBS for 1 h at RT before being incubated with a 0.3 µg/ml anti-Ki67 antibody (Novacastra). This was followed by a 1 h incubation with Alexa 488 conjugated anti-mouse antibody at RT. Keratinocyte nuclei were stained using DAPI. Images were taken on the Olympus IX-81 high content screening inverted microscope. Using a 10x objective, 7 by 11 non-overlapping quadrants were imaged. The percent of keratinocytes positive for Ki67 was determined using the Cell Profiler software^{75 (link)}.