Flow cytometric analysis was performed on cultured cells at passage 4 according to an immunofluorescence technique previously described.
24 Briefly, after trypsin digestion, cells were allowed to reestablish protein expression for 1 hour at 37°C in (2% FBS/PBS). The cells were counted, centrifuged at 1600 rpm for 5 minutes, washed in PBS, resuspended in cold 100% methanol, and permeabilized for 10 minutes at −20°C. They were centrifuged to remove the methanol, resuspended in cold 0.1% Triton X-100 (Sigma-Aldrich, Castle Hill, Australia), immediately centrifuged at 400
g for 5 minutes, diluted in 300 μL of PBS, and aliquoted into each of three tubes for direct immunofluorescence. Cells were incubated with the anti-p63 antibody for 30 minutes, washed three times in 2% BSA/PBS, blocked with human serum, incubated with a secondary FITC-conjugated Fab fragment–specific antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 20 minutes, washed three times in 2% BSA/PBS, and resuspended in 1% paraformaldehyde. Data were acquired with a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) and processed with the accompanying system software program (Cell Quest; BD Biosciences).