Analyzing Cell Proliferation in Flii Mice

Primary keratinocytes from Flii^+/−, WT and Flii^Tg/Tg mice skin were plated on glass coverslip in the well of a 6-well plate at a density of 20,000 cells in Epilife medium containing 10 μM BrdU (Sigma-Aldrich, Melbourne, Australia) as previously described [48 (link)]. Glass coverslips were pre-coated with fibronectin and collagen (50 μg/mL, Invitrogen, Australia). BrdU was allowed to incorporate into the DNA synthesis (pulse-process) for a period of 24 hr followed by washing in sterile PBS twice. New Epilife medium containing 5 μM cytochalasin D (cytD) (Sigma-Aldrich, Melbourne, Australia) was added to the cells and allowed to arrest cytokinesis (chase-process) for a period of 24 hr followed by washing in sterile PBS twice.